Methods for Reverting Methylation by Targeting Methyltransferase and Compositions Useful Therefor

ABSTRACT

Methods for restoring a desired pattern of DNA methylation, inducing re-expression of methylation-silenced tumor suppressor genes (TSGs), and/or inhibiting tumorigenicity both in vitro and in vivo in a subject in need thereof by administering an effective amount of one or more miR-29s sufficient to target one or more of DNMT3A and DNMT3B are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of Ser. No. 12/671,580filed Mar. 31, 2010, now allowed, which is a national stage applicationfiled under 35 USC §371 of international application PCT/US2008/071532filed Jul. 30, 2008, which claims the priority to U.S. ProvisionalApplication No. 60/962,795 filed Jul. 31, 2007, the entire disclosuresof which are expressly incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was not made with any Government support and theGovernment has no rights in this invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-web and is hereby incorporated by reference in itsentirety. The ASCII copy, created on Sep. 18, 2008, is named604_(—)29299_SEQ_LIST_(—)11011.txt, and is 8 MB in size.

BACKGROUND

Lung cancer is the leading cause of cancer mortality in the UnitedStates, with an incidence of approximately 213,000 new cases per yearand a very high mortality. Despite new drugs and therapeutic regimens,the prognosis for lung cancer patients has not changed significantly inthe last 20 years, emphasizing the need for novel treatment strategies.Targeting the epigenome, represents a promising therapeutic strategy incancer.

Aberrant DNA methylation has been shown to play important roles in lungcancer:

1) promoter methylation is one of the mechanisms responsible forsilencing TSGs, such as CDKN2A, CDH13, FHIT, WWOX, CDH1, and RASSF1A;

2) mRNA expression of the maintenance and de novo DNAmethyltransferases, DNMT1 and DNMT3B, respectively, were reportedlyelevated in 53% and 58% of 102 NSCLCs, respectively, and the DNMT1 mRNAlevel was shown to be an independent prognostic factor for survival;

3) DNMT1, DNMT3A and DNMT3B protein expression is elevated in lungtumors relative to normal lung tissue;

4) a specific polymorphism in the human DNMT3B promoter, whichsignificantly increases the promoter activity, has been associated withincreased lung cancer risk;

5) inhibition of DNMT1-mediated DNA methylation reduced tobaccocarcinogen-induced lung cancer in mice by >50%.

MicroRNAs (miRNAs), non-coding RNAs of 19-25 nucleotides that regulategene expression by inducing translational inhibition or cleavage oftheir target mRNAs through base pairing to partially or fullycomplementary sites, are involved in critical biological processes,including development, cell differentiation, apoptosis andproliferation. Recently, specific miRNA expression profiles, withdiagnostic and prognostic implications, have been identified forspecific cancers. Notably, members of the miR-29 family, previouslyshown to be down-regulated in NSCLC, have been predicted in silico to becomplementary to sites in the 3′ untranslated regions (3′UTRs) of DNMT3Aand B genes, using different miRNA target gene prediction algorithms(PicTar, TargetScan3.1, MiRanda, and miRGen) (FIG. 1).

Among the reported down-regulated miRNAs in lung cancer, the miR-29family (29a, 29b, and 29c), has intriguing complementarities to the 3′untranslated regions (UTRs) of DNMT3A and 3B (de novomethyltransferases), two key enzymes involved in DNA methylation, thatare frequently up-regulated in lung cancer and associated with poorprognosis.

While there is now believed that miRNAs play a role in carcinogenesis,miRNA expression is different in lung cancer versus its normalcounterpart. Further, the significance of this aberrant expression ispoorly understood.

Therefore, there is a need to determine whether miR-29s can target bothDNMT3A and DNTM3B and whether the restoration of miR-29s can normalizeaberrant patterns of methylation in lung cancers such as, for example,non-small cell lung cancer (NSCLC).

SUMMARY OF INVENTION

In one broad aspect, there is described herein a method for restoring adesired pattern of DNA methylation in a subject in need thereof,comprising administering an effective amount of one or more miR-29ssufficient to target one or more of DNMT3A and DNMT3B.

In another broad aspect, there is described herein a method for inducingre-expression of methylation-silenced tumor suppressor genes (TSGs) in asubject in need thereof, comprising administering an effective amount ofone or more miR-29s sufficient to target one or more of DNMT3A andDNMT3B. In certain embodiments, the TSG comprises one or more of FHITand WWOX.

In another broad aspect, there is described herein a method forinhibiting tumorigenicity both in vitro and in vivo in a subject in needthereof, comprising administering an effective amount of one or moremiR-29s sufficient to target one or more of DNMT3A and DNMT3B.

The methods described herein are useful in subjects suffering frommalignancies such as lung cancer.

In another aspect, there is described herein a method useful forepigenetic regulation of non-small cell lung cancer (NSCLC).

In certain embodiments, the endogenous miR-29b is useful as a primer toinitiate the retrotranscription of DNMT3B mRNA.

In another aspect, there is described herein a method for reducingglobal DNA methylation comprising administering an effective amount ofone or more miR-29s that target DNMT3A and DNMT3B, wherein expression ofthe miR-29s contribute to DNA epigenetic modifications in a cancer cell.

In another aspect, there is described herein a method for achieving DNAhypomethylation by combining at least one nucleoside analog with one ormore miR-29s sufficient to block de novo and maintenance DNMT pathways.In certain embodiments, the nucleoside analog comprises decitabine.

In another aspect, there is described herein a method for increasingexpression of a tumor suppression gene (TSG) comprising transfecting acell with one or more mi-R29s. In certain embodiments, the TSG comprisesone or more of FHIT and WWOX proteins.

In another aspect, there is described herein a method for inhibiting invitro cell growth and/or inducing apoptosis with respect to scrambledcontrols in the cells, comprising transfecting one or more cells withone or more miR-29s.

In another aspect, there is described herein a method fordown-modulating expression levels of FHIT and/or WWOX enzymes,comprising regulating the DNMT3A and/or DNMT3B by transfecting the cellwith one or more miR-29 family members. In certain embodiments, the cellis a lung cancer cell.

In another aspect, there is described herein a method for reducingglobal DNA methylation comprising inducing expression of mi-R29s in lungcancer cells.

In another aspect, there is described herein a method for restoringexpression of TSGs comprising inducing expression of mi-R29s in lungcancer cells.

In another aspect, there is described herein a method for inhibitingtumorigenicity both in vitro and in vivo comprising inducing expressionof mi-R29s in lung cancer cells.

In another aspect, there is described herein a method for developing anepigenetic therapy using synthetic miR-29s, alone or in combination withother treatments, to reactivate tumor suppressors and normalize aberrantpatterns of methylation in a cancer cell. In certain embodiments, thecancer cell is a lung cancer cell.

In another aspect, there is described herein a method of diagnosingwhether a subject has, is at risk for developing, or has a decreasesurvival prognosis for, a lung cancer-related disease, comprisingmeasuring the level of at least one miR gene product in a test samplefrom the subject; wherein an alteration in the level of the miR geneproduct in the test sample, relative to the level of a corresponding miRgene product in a control sample, is indicative of the subject eitherhaving, or being at risk for developing, the lung cancer-relateddisease; and wherein the at least one miR gene product is selected fromthe group consisting of miR-29a, miR-29b, miR-29c and combinationsthereof.

In still other aspects, there is described herein markers associatedwith a lung cancer-induced state of various cells. It has beendiscovered that the higher than normal level of expression of any ofthese markers or combination of these markers correlates with thepresence of a lung cancer-related disease in a patient. Methods areprovided for detecting the presence of a lung cancer-related disease ina sample; the absence of a in a sample; the stage of a lungcancer-related disease; and, other characteristics of a lungcancer-related disease that are relevant to the assessment, prevention,diagnosis, characterization and therapy of a lung cancer-related diseasein a patient. Methods of treating a lung cancer-related disease are alsoprovided.

In still other aspects, there is described herein methods for treating apatient afflicted with a lung cancer-related disease or at risk ofdeveloping a lung cancer-related disease. Such methods may comprisereducing the expression and/or interfering with the biological functionof a marker. In one embodiment, the method comprises providing to thepatient an antisense oligonucleotide or polynucleotide complementary toa marker nucleic acid, or a segment thereof. For example, an antisensepolynucleotide may be provided to the patient through the delivery of avector that expresses an anti-sense polynucleotide of a marker nucleicacid or a fragment thereof. In another embodiment, the method comprisesproviding to the patient an antibody, an antibody derivative or antibodyfragment, which binds specifically with a marker protein, or a fragmentof the protein.

Various objects and advantages of this invention will become apparent tothose skilled in the art from the following detailed description of thepreferred embodiment, when read in light of the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. Complementarity sites for miR-29s in the 3′UTR region of DNMT3Aand 3B.

Hsa-miR-29a [SEQ ID NO: 1]

Hsa-miR-29b [SEQ ID NO: 2]

Hsa-miR-29c [SEQ ID NO: 3]

845-869 DNMT3A [SEQ ID NO: 4]

843-869 DNMT3A [SEQ ID NO: 5]

846-869 DNMT3A [SEQ ID NO: 6]

1184-1209 DNMT3B [SEQ ID NO: 7]

244-267 DNMT3B [SEQ ID NO: 8]

1374-1398 DNMT3B [SEQ ID NO: 9]

1182-1209 DNMT3B [SEQ ID NO: 10]

1185-1209 DNMT3B [SEQ ID NO: 11

The capital and bold letters identify perfect base matches, according tothe TARGETSCAN 3.1 software. The PICTAR software identifies twoadditional match-regions between miR-29a and DNMT3B, indicated with anasterisk,*.

FIGS. 2 a-2 d). miR-29s directly target DNMT3A and B:

FIG. 2 a). Results of the luciferase assay for DNMT3s expression aftertransfection with miR-29s in A549 cells.

FIG. 2 b). Upper, assessment of expression of DNMT3A and DNMT3B mRNAs byqRT-PCR, after transfection of A549 cells with miR-29s or a negativecontrol; lower, silencing of miR-29s with antisense molecules (AS)induces increased expression of DNMT3A and DNMT3B mRNA.

FIG. 2 c). Western blot of proteins extracted from A549 cells that wereco-transfected with the GFP-repression vectors for the DNMT3A andB-3′UTR plus miR29s or scrambled oligonucleotides.

FIG. 2 d). miR-29b acts as an endogenous primer to retro-transcribe itspredicted DNMT3B mRNA target. Black font: DNMT3B cDNA (RefSeq#NM_(—)175848); blue font: cloned and sequenced cDNAs experimentallyobtained (8 clones analyzed); red font: deduced RNA sequences andcorresponding miR-29b.

3′ UTR-DNMT3B 1178-1217: [SEQ ID NO: 12]TTTAACACCTTTTACTCTTCTTAC-TGGTGCTATTTTGTAG. cDNA (8): [SEQ ID NO: 13]TTTAACACCTTTTACTCTTCTTAA:TGGTGCTA-ADAPTER.  RNA: [SEQ ID NO: 14] 3′AAAUGAGAAGAAUU:ACCACGAU 5′. Hsa-miR-29b: [SEQ ID NO: 2] 3′UUGUGACUAAAGUUUACCACGAU 5′.

Upper underlined black and blue nucleotides have no homology betweentarget and experimental cDNAs. The lower underlined red nucleotidesrepresent RNA sequence complementary to cDNAs that lack homology tomiR-29b sequence. Nucleotides in bold represent the PICTAR predictedmatch site.

FIGS. 3 a-3 d). Effect of restoration of miR-29s on the cancer cellepigenome:

FIG. 3 a). Global DNA methylation changes induced by miR-29s on A549cells harvested 48 and 72 h after transfection. The results are comparedto non-transfected cells (mock) and cells transfected with a scrambledoligonucleotide (Scr). Global DNA methylation status was determined byLC/MS-MS.

FIG. 3 b). Determination of FHIT and WWOX mRNA levels in A549 and H1299cells, 48 h after transfection with miR-29s or a negative control, byqRT PCR; miR-29s induced re-expression of FHIT and WWOX mRNAs.

FIG. 3 c). Immunoblot of FHIT and WWOX proteins in A549 and H1299 cells,72 h after transfection with miR-29s or negative control; by 72 hmiR-29s induced increased expression of FHIT and WWOX proteins. Thenumbers above the immunoblot images represent the intensity of the bandsrelative to the GAPDH gene (upper row: FHIT; lower row: WWOX).

FIG. 3 d). Graphical representation of the quantitative DNA methylationdata for FHIT and WWOX promoter region using the MassARRAY system. Eachsquare represents a single CpG or a group of CpGs analyzed, and eacharrow represents a sample. Methylation frequencies are displayed foreach experiment in a color code that extends from light green (lowermethylation frequencies) to bright red (higher methylation frequencies).

FIGS. 4 a-4 e). Effects of miR-29s on tumorigenicity of A549 cells:

FIG. 4 a). Growth curve of A549 cells transfected in vitro with miR-29s,scrambled (Scr) oligonucleotide or mock-transfected (Mock). The curvesrepresent the average cell number of 3 different experiments.

FIG. 4 b). Percent live cells were measured in A549 cells transfectedwith scrambled (Scr) oligonucleotide or with miR-29s oligonucleotides(100 nM final concentration). After 24 hours, cells were harvested andsuspended in binding buffer with annexin V-FITC and propidium iodide,followed by flow cytometry to assess cell death. Error bars indicate SD.

FIG. 4 c). Growth curve of engrafted tumors in nude mice injected withA549 cells pre-transfected (48 h before injection) with miR-29s, scroligonucleotides, or mock-transfected.

FIG. 4 d). Comparison of tumor engraftment sizes of mock-, Scr-, andmiR-29s-transfected A549 cells, 21 days post injection in nude mice. Theimages show average-sized tumors from among 5 of each category.

FIG. 4 e). Tumor weights ±SD in nude mice.

FIG. 5. DNMT3A protein expression level in NSCLCs is inverselyassociated with overall survival. Kaplan-Meier curve showing survival of172 NSCLC patients with different levels of DNMT3A expression in tumors,relative to adjacent normal lung. Patients with higher expression ofDNMT3A had shorter overall survival (P=0.029). There was a trend towarda similar association of DNMT3B protein expression level with survivalbut no such association for DNMT1.

FIG. 6. Correlation of endogenous miR-29 levels with DNMT3A/B mRNAlevels. Inverse correlation between endogenous mRNA levels of DNMT3A, 3Band endogenous levels of miR-29s, determined by qRT PCR in 14 NSCLCs.R=regression coefficient. □=regression line; ♦=actual samplecorrelations.

DESCRIPTION OF EMBODIMENTS

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Thedisclosures of these publications, patents and published patentspecifications are hereby incorporated by reference into the presentdisclosure to more fully describe the state of the art to which thisinvention pertains.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art (e.g., in cell culture, molecular genetics, nucleic acidchemistry, hybridization techniques and biochemistry). Standardtechniques are used for molecular, genetic and biochemical methods whichare within the skill of the art. Such techniques are explained fully inthe literature. See, for example, Molecular Cloning A Laboratory Manual,2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press: 1989); DNA Cloning, Volumes I and II (Glover ed.,1985); Oligonucleotide Synthesis (Gait ed., 1984); Mullis et al. U.S.Pat. No. 4,683,195; Nucleic Acid Hybridization (Hames & Higgins eds.,1984); Transcription And Translation (Hames & Higgins eds., 1984);Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987);Immobilized Cells And Enzymes (IRL Press, 1986); Perbal, A PracticalGuide To Molecular Cloning (1984); the treatise, Methods In Enzymology(Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells(Miller and Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (Weirand Blackwell, eds., 1986); The Laboratory Rat, editor in chief: Mark A.Suckow; authors: Sharp and LaRegina. CRC Press, Boston, 1988, which areincorporated herein by reference) and chemical methods.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

As used herein interchangeably, a “miR gene product,” “microRNA,” “miR,”or “miRNA” refers to the unprocessed (e.g., precursor) or processed(e.g., mature) RNA transcript from a miR gene. As the miR gene productsare not translated into protein, the term “miR gene products” does notinclude proteins. The unprocessed miR gene transcript is also called a“miR precursor” or “miR prec” and typically comprises an RNA transcriptof about 70-100 nucleotides in length. The miR precursor can beprocessed by digestion with an RNAse (for example, Dicer, Argonaut, orRNAse III (e.g., E. coli RNAse III)) into an active 19-25 nucleotide RNAmolecule. This active 19-25 nucleotide RNA molecule is also called the“processed” miR gene transcript or “mature” miRNA.

A “marker” is a gene or protein whose altered level of expression in atissue or cell from its expression level in normal or healthy tissue orcell is associated with a disease state.

The “normal” level of expression of a marker is the level of expressionof the marker in lung cells of a human subject or patient not afflictedwith a lung cancer-related disease.

An “over-expression” or “significantly higher level of expression” of amarker refers to an expression level in a test sample that is greaterthan the standard error of the assay employed to assess expression, andin certain embodiments, at least twice, and in other embodiments, three,four, five or ten times the expression level of the marker in a controlsample (e.g., sample from a healthy subject not having the markerassociated disease) and in certain embodiments, the average expressionlevel of the marker in several control samples.

A “significantly lower level of expression” of a marker refers to anexpression level in a test sample that is at least twice, and in certainembodiments, three, four, five or ten times lower than the expressionlevel of the marker in a control sample (e.g., sample from a healthysubject not having the marker associated disease) and in certainembodiments, the average expression level of the marker in severalcontrol samples.

A “kit” is any manufacture (e.g. a package or container) comprising atleast one reagent, e.g., a probe, for specifically detecting theexpression of a marker. The kit may be promoted, distributed or sold asa unit for performing the methods of the present invention.

“Proteins” encompass marker proteins and their fragments; variant markerproteins and their fragments; peptides and polypeptides comprising an atleast 15 amino acid segment of a marker or variant marker protein; andfusion proteins comprising a marker or variant marker protein, or an atleast 15 amino acid segment of a marker or variant marker protein.

In a first broad aspect, there is provided herein the identification ofparticular microRNAs whose expression is altered in cancer cellsassociated with different lung cancers, relative to normal controlcells.

The active 19-25 nucleotide RNA molecule can be obtained from the miRprecursor through natural processing routes (e.g., using intact cells orcell lysates) or by synthetic processing routes (e.g., using isolatedprocessing enzymes, such as isolated Dicer, Argonaut, or RNAse III). Itis understood that the active 19-25 nucleotide RNA molecule can also beproduced directly by biological or chemical synthesis, without havingbeen processed from the miR precursor. When a microRNA is referred toherein by name, the name corresponds to both the precursor and matureforms, unless otherwise indicated.

In one aspect, there is provided herein methods of diagnosing whether asubject has, or is at risk for developing, a lung cancer, comprisingmeasuring the level of at least one miR gene product in a test samplefrom the subject and comparing the level of the miR gene product in thetest sample to the level of a corresponding miR gene product in acontrol sample. As used herein, a “subject” can be any mammal that has,or is suspected of having, a lung cancer. In a preferred embodiment, thesubject is a human who has, or is suspected of having, a lung cancer.

In one embodiment, the at least one miR gene product measured in thetest sample is selected from the group consisting of miR-29a, miR-29b,miR-29c, and combinations thereof. In a particular embodiment, the miRgene product is miR-29b.

The lung cancer-related disease can be any disorder or cancer thatarises from the lung tissues. Such cancers are typically associated withthe formation and/or presence of tumor masses and can be, for example,any form of lung cancer, for example, lung cancers of differinghistology (e.g., adenocarcinoma, squamous cell carcinoma). Furthermore,the lung cancer may be associated with a particular prognosis (e.g., lowsurvival rate, fast progression).

The level of at least one miR gene product can be measured in abiological sample (e.g., cells, tissues) obtained from the subject. Forexample, a tissue sample (e.g., from a tumor) can be removed from asubject suspected of having a lung cancer-related disease byconventional biopsy techniques. In another embodiment, a blood samplecan be removed from the subject, and blood cells (e.g., white bloodcells) can be isolated for DNA extraction by standard techniques. Theblood or tissue sample is preferably obtained from the subject prior toinitiation of radiotherapy, chemotherapy or other therapeutic treatment.A corresponding control tissue or blood sample can be obtained fromunaffected tissues of the subject, from a normal human individual orpopulation of normal individuals, or from cultured cells correspondingto the majority of cells in the subject's sample. The control tissue orblood sample is then processed along with the sample from the subject,so that the levels of miR gene product produced from a given miR gene incells from the subject's sample can be compared to the corresponding miRgene product levels from cells of the control sample. A reference miRexpression standard for the biological sample can also be used as acontrol.

An alteration (e.g., an increase or decrease) in the level of a miR geneproduct in the sample obtained from the subject, relative to the levelof a corresponding miR gene product in a control sample, is indicativeof the presence of a lung cancer-related disease in the subject.

In one embodiment, the level of the at least one miR gene product in thetest sample is greater than the level of the corresponding miR geneproduct in the control sample (i.e., expression of the miR gene productis “up-regulated”). As used herein, expression of a miR gene product is“up-regulated” when the amount of miR gene product in a cell or tissuesample from a subject is greater than the amount of the same geneproduct in a control cell or tissue sample.

In another embodiment, the level of the at least one miR gene product inthe test sample is less than the level of the corresponding miR geneproduct in the control sample (i.e., expression of the miR gene productis “down-regulated”). As used herein, expression of a miR gene is“down-regulated” when the amount of miR gene product produced from thatgene in a cell or tissue sample from a subject is less than the amountproduced from the same gene in a control cell or tissue sample.

The relative miR gene expression in the control and normal samples canbe determined with respect to one or more RNA expression standards. Thestandards can comprise, for example, a zero miR gene expression level,the miR gene expression level in a standard cell line, the miR geneexpression level in unaffected tissues of the subject, or the averagelevel of miR gene expression previously obtained for a population ofnormal human controls.

The level of a miR gene product in a sample can be measured using anytechnique that is suitable for detecting RNA expression levels in abiological sample. Suitable techniques (e.g., Northern blot analysis,RT-PCR, in situ hybridization) for determining RNA expression levels ina biological sample (e.g., cells, tissues) are well known to those ofskill in the art. In a particular embodiment, the level of at least onemiR gene product is detected using Northern blot analysis. For example,total cellular RNA can be purified from cells by homogenization in thepresence of nucleic acid extraction buffer, followed by centrifugation.Nucleic acids are precipitated, and DNA is removed by treatment withDNase and precipitation. The RNA molecules are then separated by gelelectrophoresis on agarose gels according to standard techniques, andtransferred to nitrocellulose filters. The RNA is then immobilized onthe filters by heating. Detection and quantification of specific RNA isaccomplished using appropriately labeled DNA or RNA probes complementaryto the RNA in question. See, for example, Molecular Cloning: ALaboratory Manual, J. Sambrook et al., eds., 2nd edition, Cold SpringHarbor Laboratory Press, 1989, Chapter 7, the entire disclosure of whichis incorporated by reference.

Suitable probes for Northern blot hybridization of a given miR geneproduct can be produced from the nucleic acid sequences and include, butare not limited to, probes having at least about 70%, 75%, 80%, 85%,90%, 95%, 98%, 99% or complete complementarity to a miR gene product ofinterest. Methods for preparation of labeled DNA and RNA probes, and theconditions for hybridization thereof to target nucleotide sequences, aredescribed in Molecular Cloning: A Laboratory Manual, J. Sambrook et al.,eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapters10 and 11, the disclosures of which are incorporated herein byreference.

In one non-limiting example, the nucleic acid probe can be labeled with,e.g., a radionuclide, such as ³H, ³²P, ³³P, ¹⁴C, or ³⁵S; a heavy metal;a ligand capable of functioning as a specific binding pair member for alabeled ligand (e.g., biotin, avidin or an antibody); a fluorescentmolecule; a chemiluminescent molecule; an enzyme or the like.

Probes can be labeled to high specific activity by either the nicktranslation method of Rigby et al. (1977), J. Mol. Biol. 113:237-251 orby the random priming method of Fienberg et al. (1983), Anal. Biochem.132:6-13, the entire disclosures of which are incorporated herein byreference. The latter is the method of choice for synthesizing³²P-labeled probes of high specific activity from single-stranded DNA orfrom RNA templates. For example, by replacing preexisting nucleotideswith highly radioactive nucleotides according to the nick translationmethod, it is possible to prepare ³²P-labeled nucleic acid probes with aspecific activity well in excess of 10⁸ cpm/microgram.

Autoradiographic detection of hybridization can then be performed byexposing hybridized filters to photographic film. Densitometric scanningof the photographic films exposed by the hybridized filters provides anaccurate measurement of miR gene transcript levels. Using anotherapproach, miR gene transcript levels can be quantified by computerizedimaging systems, such as the Molecular Dynamics 400-B 2D Phosphorimageravailable from Amersham Biosciences, Piscataway, N.J.

Where radionuclide labeling of DNA or RNA probes is not practical, therandom-primer method can be used to incorporate an analogue, forexample, the dTTP analogue5-(N—(N-biotinyl-epsilon-aminocaproyl)-3-aminoallyl)deoxyuridinetriphosphate, into the probe molecule. The biotinylated probeoligonucleotide can be detected by reaction with biotin-bindingproteins, such as avidin, streptavidin, and antibodies (e.g.,anti-biotin antibodies) coupled to fluorescent dyes or enzymes thatproduce color reactions.

In addition to Northern and other RNA hybridization techniques,determining the levels of RNA transcripts can be accomplished using thetechnique of in situ hybridization. This technique requires fewer cellsthan the Northern blotting technique, and involves depositing wholecells onto a microscope cover slip and probing the nucleic acid contentof the cell with a solution containing radioactive or otherwise labelednucleic acid (e.g., cDNA or RNA) probes. This technique is particularlywell-suited for analyzing tissue biopsy samples from subjects. Thepractice of the in situ hybridization technique is described in moredetail in U.S. Pat. No. 5,427,916, the entire disclosure of which isincorporated herein by reference.

In one non-limiting example, suitable probes for in situ hybridizationof a given miR gene product can be produced from the nucleic acidsequences, and include, but are not limited to, probes having at leastabout 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or complete complementarityto a miR gene product of interest, as described above.

The relative number of miR gene transcripts in cells can also bedetermined by reverse transcription of miR gene transcripts, followed byamplification of the reverse-transcribed transcripts by polymerase chainreaction (RT-PCR). The levels of miR gene transcripts can be quantifiedin comparison with an internal standard, for example, the level of mRNAfrom a “housekeeping” gene present in the same sample. A suitable“housekeeping” gene for use as an internal standard includes, e.g.,myosin or glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Methods forperforming quantitative and semi-quantitative RT-PCR, and variationsthereof, are well known to those of skill in the art.

In some instances, it may be desirable to simultaneously determine theexpression level of a plurality of different miR gene products in asample. In other instances, it may be desirable to determine theexpression level of the transcripts of all known miR genes correlatedwith a cancer. Assessing cancer-specific expression levels for hundredsof miR genes or gene products is time consuming and requires a largeamount of total RNA (e.g., at least 20 μg for each Northern blot) andautoradiographic techniques that require radioactive isotopes.

To overcome these limitations, an oligolibrary, in microchip format(i.e., a microarray), may be constructed containing a set ofoligonucleotide (e.g., oligodeoxynucleotides) probes that are specificfor a set of miR genes. Using such a microarray, the expression level ofmultiple microRNAs in a biological sample can be determined by reversetranscribing the RNAs to generate a set of target oligodeoxynucleotides,and hybridizing them to probe the oligonucleotides on the microarray togenerate a hybridization, or expression, profile. The hybridizationprofile of the test sample can then be compared to that of a controlsample to determine which microRNAs have an altered expression level inlung cancer cells.

As used herein, “probe oligonucleotide” or “probe oligodeoxynucleotide”refers to an oligonucleotide that is capable of hybridizing to a targetoligonucleotide. “Target oligonucleotide” or “targetoligodeoxynucleotide” refers to a molecule to be detected (e.g., viahybridization). By “miR-specific probe oligonucleotide” or “probeoligonucleotide specific for a miR” is meant a probe oligonucleotidethat has a sequence selected to hybridize to a specific miR geneproduct, or to a reverse transcript of the specific miR gene product.

An “expression profile” or “hybridization profile” of a particularsample is essentially a fingerprint of the state of the sample; whiletwo states may have any particular gene similarly expressed, theevaluation of a number of genes simultaneously allows the generation ofa gene expression profile that is unique to the state of the cell. Thatis, normal tissue may be distinguished from cancerous (e.g., tumor)tissue, and within cancerous tissue, different prognosis states (forexample, good or poor long term survival prospects) may be determined Bycomparing expression profiles of the cancer tissue in different states,information regarding which genes are important (including both up- anddown-regulation of genes) in each of these states is obtained. Theidentification of sequences that are differentially expressed in cancertissue, as well as differential expression resulting in differentprognostic outcomes, allows the use of this information in a number ofways.

In one non-limiting example, a particular treatment regime may beevaluated (e.g., to determine whether a chemotherapeutic drug acts toimprove the long-term prognosis in a particular patient) Similarly,diagnosis may be done or confirmed by comparing patient samples withknown expression profiles. Furthermore, these gene expression profiles(or individual genes) allow screening of drug candidates that suppressthe lung cancer expression profile or convert a poor prognosis profileto a better prognosis profile.

Accordingly, there is also provided herein methods of diagnosing whethera subject has, or is at risk for developing, a lung cancer, comprisingreverse transcribing RNA from a test sample obtained from the subject toprovide a set of target oligodeoxynucleotides, hybridizing the targetoligodeoxynucleotides to a microarray comprising miRNA-specific probeoligonucleotides to provide a hybridization profile for the test sample,and comparing the test sample hybridization profile to a hybridizationprofile generated from a control sample or reference standard, whereinan alteration in the signal of at least one miRNA is indicative of thesubject either having, or being at risk for developing, lung cancer.

In one embodiment, the microarray comprises miRNA-specific probeoligonucleotides for a substantial portion of all known human miRNAs. Ina particular embodiment, the microarray comprises miRNA-specific probeoligonucleotides for one or more miRNAs selected from the groupconsisting of miR-29a, miR-29b, miR-29c and combinations thereof.

The microarray can be prepared from gene-specific oligonucleotide probesgenerated from known miRNA sequences. The array may contain twodifferent oligonucleotide probes for each miRNA, one containing theactive, mature sequence and the other being specific for the precursorof the miRNA. The array may also contain controls, such as one or moremouse sequences differing from human orthologs by only a few bases,which can serve as controls for hybridization stringency conditions.tRNAs or other RNAs (e.g., rRNAs, mRNAs) from both species may also beprinted on the microchip, providing an internal, relatively stable,positive control for specific hybridization. One or more appropriatecontrols for non-specific hybridization may also be included on themicrochip. For this purpose, sequences are selected based upon theabsence of any homology with any known miRNAs.

The microarray may be fabricated using techniques known in the art. Forexample, probe oligonucleotides of an appropriate length, e.g., 40nucleotides, are 5′-amine modified at position C6 and printed usingcommercially available microarray systems, e.g., the GeneMachineOmniGrid™ 100 Microarrayer and Amersham CodeLink™ activated slides.Labeled cDNA oligomer corresponding to the target RNAs is prepared byreverse transcribing the target RNA with labeled primer. Following firststrand synthesis, the RNA/DNA hybrids are denatured to degrade the RNAtemplates. The labeled target cDNAs thus prepared are then hybridized tothe microarray chip under hybridizing conditions, e.g., 6×SSPE/30%formamide at 25° C. for 18 hours, followed by washing in 0.75×TNT (TrisHCl/NaCl/Tween 20) at 37° C. for 40 minutes. At positions on the arraywhere the immobilized probe DNA recognizes a complementary target cDNAin the sample, hybridization occurs. The labeled target cDNA marks theexact position on the array where binding occurs, allowing automaticdetection and quantification. The output consists of a list ofhybridization events, indicating the relative abundance of specific cDNAsequences, and therefore the relative abundance of the correspondingcomplementary miRs, in the patient sample.

According to one embodiment, the labeled cDNA oligomer is abiotin-labeled cDNA, prepared from a biotin-labeled primer. Themicroarray is then processed by direct detection of thebiotin-containing transcripts using, e.g., Streptavidin-Alexa647conjugate, and scanned utilizing conventional scanning methods. Imageintensities of each spot on the array are proportional to the abundanceof the corresponding miR in the patient sample.

The use of the array has several advantages for miRNA expressiondetection. First, the global expression of several hundred genes can beidentified in the same sample at one time point. Second, through carefuldesign of the oligonucleotide probes, expression of both mature andprecursor molecules can be identified. Third, in comparison withNorthern blot analysis, the chip requires a small amount of RNA, andprovides reproducible results using 2.5 μg of total RNA. The relativelylimited number of miRNAs (a few hundred per species) allows theconstruction of a common microarray for several species, with distinctoligonucleotide probes for each. Such a tool allows for analysis oftrans-species expression for each known miR under various conditions.

In addition to use for quantitative expression level assays of specificmiRs, a microchip containing miRNA-specific probe oligonucleotidescorresponding to a substantial portion of the miRNome, preferably theentire miRNome, may be employed to carry out miR gene expressionprofiling, for analysis of miR expression patterns. Distinct miRsignatures can be associated with established disease markers, ordirectly with a disease state.

According to the expression profiling methods described herein, totalRNA from a sample from a subject suspected of having a lungcancer-related disease quantitatively reverse transcribed to provide aset of labeled target oligodeoxynucleotides complementary to the RNA inthe sample. The target oligodeoxynucleotides are then hybridized to amicroarray comprising miRNA-specific probe oligonucleotides to provide ahybridization profile for the sample. The result is a hybridizationprofile for the sample representing the expression pattern of miRNA inthe sample. The hybridization profile comprises the signal from thebinding of the target oligodeoxynucleotides from the sample to themiRNA-specific probe oligonucleotides in the microarray. The profile maybe recorded as the presence or absence of binding (signal vs. zerosignal).

More preferably, the profile recorded includes the intensity of thesignal from each hybridization. The profile is compared to thehybridization profile generated from a normal, i.e., noncancerous,control sample. An alteration in the signal is indicative of thepresence of, or propensity to develop, cancer in the subject.

Other techniques for measuring miR gene expression are also within theskill in the art, and include various techniques for measuring rates ofRNA transcription and degradation.

There is also provided herein methods of determining the prognosis of asubject with a lung cancer, comprising measuring the level of at leastone miR gene product, which is associated with a particular prognosis ina lung cancer-related disease (e.g., a good or positive prognosis, apoor or adverse prognosis), in a test sample from the subject.

According to these methods, an alteration in the level of a miR geneproduct that is associated with a particular prognosis in the testsample, as compared to the level of a corresponding miR gene product ina control sample, is indicative of the subject having a lung cancer witha particular prognosis. In one embodiment, the miR gene product isassociated with an adverse (i.e., poor) prognosis. Examples of anadverse prognosis include, but are not limited to, low survival rate andrapid disease progression. In certain embodiments, the level of the atleast one miR gene product is measured by reverse transcribing RNA froma test sample obtained from the subject to provide a set of targetoligodeoxynucleotides, hybridizing the target oligodeoxynucleotides to amicroarray that comprises miRNA-specific probe oligonucleotides toprovide a hybridization profile for the test sample, and comparing thetest sample hybridization profile to a hybridization profile generatedfrom a control sample.

Without wishing to be bound by any one theory, it is believed thatalterations in the level of one or more miR gene products in cells canresult in the deregulation of one or more intended targets for thesemiRs, which can lead to the formation of lung cancers. Therefore,altering the level of the miR gene product (e.g., by decreasing thelevel of a miR gene product that is up-regulated in lung cancer cells,by increasing the level of a miR gene product that is down-regulated inlung cancer cells) may successfully treat the lung cancer.

Accordingly, there is further provided herein methods of inhibitingtumorigenesis in a subject who has, or is suspected of having, a lungcancer wherein at least one miR gene product is deregulated (e.g.,down-regulated, up-regulated) in the cancer cells of the subject. Whenthe at least one isolated miR gene product is down-regulated in thecancer cells (e.g., miR-29 family), the method comprises administeringan effective amount of the at least one isolated miR gene product, or anisolated variant or biologically-active fragment thereof, such thatproliferation of cancer cells in the subject is inhibited.

For example, when a miR gene product is down-regulated in a cancer cellin a subject, administering an effective amount of an isolated miR geneproduct to the subject can inhibit proliferation of the cancer cell. Theisolated miR gene product that is administered to the subject can beidentical to the endogenous wild-type miR gene product (e.g., a miR geneproduct) that is down-regulated in the cancer cell or it can be avariant or biologically-active fragment thereof.

As defined herein, a “variant” of a miR gene product refers to a miRNAthat has less than 100% identity to a corresponding wild-type miR geneproduct and possesses one or more biological activities of thecorresponding wild-type miR gene product. Examples of such biologicalactivities include, but are not limited to, inhibition of expression ofa target RNA molecule (e.g., inhibiting translation of a target RNAmolecule, modulating the stability of a target RNA molecule, inhibitingprocessing of a target RNA molecule) and inhibition of a cellularprocess associated with lung cancer (e.g., cell differentiation, cellgrowth, cell death). These variants include species variants andvariants that are the consequence of one or more mutations (e.g., asubstitution, a deletion, an insertion) in a miR gene. In certainembodiments, the variant is at least about 70%, 75%, 80%, 85%, 90%, 95%,98%, or 99% identical to a corresponding wild-type miR gene product.

As defined herein, a “biologically-active fragment” of a miR geneproduct refers to an RNA fragment of a miR gene product that possessesone or more biological activities of a corresponding wild-type miR geneproduct. As described above, examples of such biological activitiesinclude, but are not limited to, inhibition of expression of a targetRNA molecule and inhibition of a cellular process associated with a lungcancer. In certain embodiments, the biologically-active fragment is atleast about 5, 7, 10, 12, 15, or 17 nucleotides in length.

In a particular embodiment, an isolated miR gene product can beadministered to a subject in combination with one or more additionalanti-cancer treatments. Suitable anti-cancer treatments include, but arenot limited to, chemotherapy, radiation therapy and combinations thereof(e.g., chemoradiation).

When the at least one isolated miR gene product is up-regulated in thecancer cells, the method comprises administering to the subject aneffective amount of at least one compound for inhibiting expression ofthe at least one miR gene product, referred to herein as miR geneexpression-inhibition compounds, such that proliferation of the cancercells is inhibited. In a particular embodiment, the at least one miRexpression-inhibition compound is specific for a miR gene productselected from the group consisting miR-29 family, including miR-29a,miR-29b, miR-29c, and combinations thereof.

The terms “treat”, “treating” and “treatment”, as used herein, refer toameliorating symptoms associated with a disease or condition, forexample, a lung cancer, including preventing or delaying the onset ofthe disease symptoms, and/or lessening the severity or frequency ofsymptoms of the disease or condition. The terms “subject”, “patient” and“individual” are defined herein to include animals, such as mammals,including, but not limited to, primates, cows, sheep, goats, horses,dogs, cats, rabbits, guinea pigs, rats, mice or other bovine, ovine,equine, canine, feline, rodent, or murine species. In a preferredembodiment, the animal is a human.

As used herein, an “effective amount” of an isolated miR gene product isan amount sufficient to inhibit proliferation of a cancer cell in asubject suffering from a lung cancer. One skilled in the art can readilydetermine an effective amount of a miR gene product to be administeredto a given subject, by taking into account factors, such as the size andweight of the subject; the extent of disease penetration; the age,health and sex of the subject; the route of administration; and whetherthe administration is regional or systemic.

For example, an effective amount of an isolated miR gene product can bebased on the approximate weight of a tumor mass to be treated. Theapproximate weight of a tumor mass can be determined by calculating theapproximate volume of the mass, wherein one cubic centimeter of volumeis roughly equivalent to one gram. An effective amount of the isolatedmiR gene product based on the weight of a tumor mass can be in the rangeof about 10-500 micrograms/gram of tumor mass. In certain embodiments,the tumor mass can be at least about 10 micrograms/gram of tumor mass,at least about 60 micrograms/gram of tumor mass or at least about 100micrograms/gram of tumor mass.

An effective amount of an isolated miR gene product can also be based onthe approximate or estimated body weight of a subject to be treated.Preferably, such effective amounts are administered parenterally orenterally, as described herein. For example, an effective amount of theisolated miR gene product is administered to a subject can range fromabout 5μ to about 3000 micrograms/kg of body weight, from about 700-1000micrograms/kg of body weight, or greater than about 1000 micrograms/kgof body weight.

One skilled in the art can also readily determine an appropriate dosageregimen for the administration of an isolated miR gene product to agiven subject. For example, a miR gene product can be administered tothe subject once (e.g., as a single injection or deposition).Alternatively, a miR gene product can be administered once or twicedaily to a subject for a period of from about three to abouttwenty-eight days, more particularly from about seven to about ten days.In a particular dosage regimen, a miR gene product is administered oncea day for seven days. Where a dosage regimen comprises multipleadministrations, it is understood that the effective amount of the miRgene product administered to the subject can comprise the total amountof gene product administered over the entire dosage regimen.

As used herein, an “isolated” miR gene product is one that issynthesized, or altered or removed from the natural state through humanintervention. For example, a synthetic miR gene product, or a miR geneproduct partially or completely separated from the coexisting materialsof its natural state, is considered to be “isolated.” An isolated miRgene product can exist in substantially-purified form, or can exist in acell into which the miR gene product has been delivered. Thus, a miRgene product that is deliberately delivered to, or expressed in, a cellis considered an “isolated” miR gene product. A miR gene productproduced inside a cell from a miR precursor molecule is also consideredto be an “isolated” molecule. According to one particular embodiment,the isolated miR gene products described herein can be used for themanufacture of a medicament for treating a lung cancer in a subject(e.g., a human).

Isolated miR gene products can be obtained using a number of standardtechniques. For example, the miR gene products can be chemicallysynthesized or recombinantly produced using methods known in the art. Inone embodiment, miR gene products are chemically synthesized usingappropriately protected ribonucleoside phosphoramidites and aconventional DNA/RNA synthesizer. Commercial suppliers of synthetic RNAmolecules or synthesis reagents include, e.g., Proligo (Hamburg,Germany), Dharmacon Research (Lafayette, Colo., U.S.A.), Pierce Chemical(part of Perbio Science, Rockford, Ill., U.S.A.), Glen Research(Sterling, Va., U.S.A.), ChemGenes (Ashland, Mass., U.S.A.) and Cruachem(Glasgow, UK).

Alternatively, the miR gene products can be expressed from recombinantcircular or linear DNA plasmids using any suitable promoter. Suitablepromoters for expressing RNA from a plasmid include, e.g., the U6 or H1RNA pol III promoter sequences, or the cytomegalovirus promoters.Selection of other suitable promoters is within the skill in the art.The recombinant plasmids of the invention can also comprise inducible orregulatable promoters for expression of the miR gene products in cancercells.

The miR gene products that are expressed from recombinant plasmids canbe isolated from cultured cell expression systems by standardtechniques. The miR gene products that are expressed from recombinantplasmids can also be delivered to, and expressed directly in, the cancercells. The use of recombinant plasmids to deliver the miR gene productsto cancer cells is discussed in more detail below.

The miR gene products can be expressed from a separate recombinantplasmid, or they can be expressed from the same recombinant plasmid. Inone embodiment, the miR gene products are expressed as RNA precursormolecules from a single plasmid, and the precursor molecules areprocessed into the functional miR gene product by a suitable processingsystem, including, but not limited to, processing systems extant withina cancer cell. Other suitable processing systems include, e.g., the invitro Drosophila cell lysate system (e.g., as described in U.S.Published Patent Application No. 2002/0086356 to Tuschl et al., theentire disclosure of which is incorporated herein by reference) and theE. coli RNAse III system (e.g., as described in U.S. Published PatentApplication No. 2004/0014113 to Yang et al., the entire disclosure ofwhich is incorporated herein by reference).

Selection of plasmids suitable for expressing the miR gene products,methods for inserting nucleic acid sequences into the plasmid to expressthe gene products, and methods of delivering the recombinant plasmid tothe cells of interest are within the skill in the art. See, for example,Zeng et al. (2002), Molecular Cell 9:1327-1333; Tuschl (2002), Nat.Biotechnol, 20:446-448; Brummelkamp et al. (2002), Science 296:550-553;Miyagishi et al. (2002), Nat. Biotechnol. 20:497-500; Paddison et al.(2002), Genes Dev. 16:948-958; Lee et al. (2002), Nat. Biotechnol.20:500-505; and Paul et al. (2002), Nat. Biotechnol. 20:505-508, theentire disclosures of which are incorporated herein by reference.

In one embodiment, a plasmid expressing the miR gene products comprisesa sequence encoding a miR precursor RNA under the control of the CMVintermediate-early promoter. As used herein, “under the control” of apromoter means that the nucleic acid sequences encoding the miR geneproduct are located 3′ of the promoter, so that the promoter caninitiate transcription of the miR gene product coding sequences.

The miR gene products can also be expressed from recombinant viralvectors. It is contemplated that the miR gene products can be expressedfrom two separate recombinant viral vectors, or from the same viralvector. The RNA expressed from the recombinant viral vectors can eitherbe isolated from cultured cell expression systems by standardtechniques, or can be expressed directly in cancer cells. The use ofrecombinant viral vectors to deliver the miR gene products to cancercells is discussed in more detail below.

The recombinant viral vectors of the invention comprise sequencesencoding the miR gene products and any suitable promoter for expressingthe RNA sequences. Suitable promoters include, but are not limited to,the U6 or H1 RNA pol III promoter sequences, or the cytomegaloviruspromoters. Selection of other suitable promoters is within the skill inthe art. The recombinant viral vectors of the invention can alsocomprise inducible or regulatable promoters for expression of the miRgene products in a cancer cell.

Any viral vector capable of accepting the coding sequences for the miRgene products can be used; for example, vectors derived from adenovirus(AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses(LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like.The tropism of the viral vectors can be modified by pseudotyping thevectors with envelope proteins or other surface antigens from otherviruses, or by substituting different viral capsid proteins, asappropriate.

For example, lentiviral vectors of the invention can be pseudotyped withsurface proteins from vesicular stomatitis virus (VSV), rabies, Ebola,Mokola, and the like. AAV vectors of the invention can be made to targetdifferent cells by engineering the vectors to express different capsidprotein serotypes. For example, an AAV vector expressing a serotype 2capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsidgene in the AAV 2/2 vector can be replaced by a serotype 5 capsid geneto produce an AAV 2/5 vector. Techniques for constructing AAV vectorsthat express different capsid protein serotypes are within the skill inthe art; see, e.g., Rabinowitz, I. E., et al. (2002), J. Virol.76:791-801, the entire disclosure of which is incorporated herein byreference.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingRNA into the vector, methods of delivering the viral vector to the cellsof interest, and recovery of the expressed RNA products are within theskill in the art. See, for example, Dornburg (1995), Gene Therapy2:301-310; Eglitis (1988), Biotechniques 6:608-614; Miller (1990), Hum.Gene Therapy 1:5-14; and Anderson (1998), Nature 392:25-30, the entiredisclosures of which are incorporated herein by reference.

Particularly suitable viral vectors are those derived from AV and AAV. Asuitable AV vector for expressing the miR gene products, a method forconstructing the recombinant AV vector, and a method for delivering thevector into target cells, are described in Xia et al. (2002), Nat.Biotech. 20:1006-1010, the entire disclosure of which is incorporatedherein by reference. Suitable AAV vectors for expressing the miR geneproducts, methods for constructing the recombinant AAV vector, andmethods for delivering the vectors into target cells are described inSamulski et al. (1987), J. Virol. 61:3096-3101; Fisher et al. (1996), J.Virol., 70:520-532; Samulski et al. (1989), J. Virol. 63:3822-3826; U.S.Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International PatentApplication No. WO 94/13788; and International Patent Application No. WO93/24641, the entire disclosures of which are incorporated herein byreference. In one embodiment, the miR gene products are expressed from asingle recombinant AAV vector comprising the CMV intermediate earlypromoter.

In a certain embodiment, a recombinant AAV viral vector of the inventioncomprises a nucleic acid sequence encoding a miR precursor RNA inoperable connection with a polyT termination sequence under the controlof a human U6 RNA promoter. As used herein, “in operable connection witha polyT termination sequence” means that the nucleic acid sequencesencoding the sense or antisense strands are immediately adjacent to thepolyT termination signal in the 5′ direction. During transcription ofthe miR sequences from the vector, the polyT termination signals act toterminate transcription.

In other embodiments of the treatment methods of the invention, aneffective amount of at least one compound that inhibits miR expressioncan be administered to the subject. As used herein, “inhibiting miRexpression” means that the production of the precursor and/or active,mature form of miR gene product after treatment is less than the amountproduced prior to treatment. One skilled in the art can readilydetermine whether miR expression has been inhibited in a cancer cell,using, for example, the techniques for determining miR transcript leveldiscussed above for the diagnostic method. Inhibition can occur at thelevel of gene expression (i.e., by inhibiting transcription of a miRgene encoding the miR gene product) or at the level of processing (e.g.,by inhibiting processing of a miR precursor into a mature, active miR).

As used herein, an “effective amount” of a compound that inhibits miRexpression is an amount sufficient to inhibit proliferation of a cancercell in a subject suffering from a cancer (e.g., a lung cancer). Oneskilled in the art can readily determine an effective amount of a miRexpression-inhibition compound to be administered to a given subject, bytaking into account factors, such as the size and weight of the subject;the extent of disease penetration; the age, health and sex of thesubject; the route of administration; and whether the administration isregional or systemic.

For example, an effective amount of the expression-inhibition compoundcan be based on the approximate weight of a tumor mass to be treated, asdescribed herein. An effective amount of a compound that inhibits miRexpression can also be based on the approximate or estimated body weightof a subject to be treated, as described herein.

One skilled in the art can also readily determine an appropriate dosageregimen for administering a compound that inhibits miR expression to agiven subject.

Suitable compounds for inhibiting miR gene expression includedouble-stranded RNA (such as short- or small-interfering RNA or“siRNA”), antisense nucleic acids, and enzymatic RNA molecules, such asribozymes. Each of these compounds can be targeted to a given miR geneproduct and interfere with the expression of (e.g., inhibit translationof, induce cleavage or destruction of) the target miR gene product.

For example, expression of a given miR gene can be inhibited by inducingRNA interference of the miR gene with an isolated double-stranded RNA(“dsRNA”) molecule which has at least 90%, for example at least 95%, atleast 98%, at least 99%, or 100%, sequence homology with at least aportion of the miR gene product. In a particular embodiment, the dsRNAmolecule is a “short or small interfering RNA” or “siRNA.”

siRNA useful in the present methods comprise short double-stranded RNAfrom about 17 nucleotides to about 29 nucleotides in length, preferablyfrom about 19 to about 25 nucleotides in length. The siRNA comprise asense RNA strand and a complementary antisense RNA strand annealedtogether by standard Watson-Crick base-pairing interactions (hereinafter“base-paired”). The sense strand comprises a nucleic acid sequence thatis substantially identical to a nucleic acid sequence contained withinthe target miR gene product.

As used herein, a nucleic acid sequence in an siRNA which is“substantially identical” to a target sequence contained within thetarget mRNA is a nucleic acid sequence that is identical to the targetsequence, or that differs from the target sequence by one or twonucleotides. The sense and antisense strands of the siRNA can comprisetwo complementary, single-stranded RNA molecules, or can comprise asingle molecule in which two complementary portions are base-paired andare covalently linked by a single-stranded “hairpin” area.

The siRNA can also be altered RNA that differs from naturally-occurringRNA by the addition, deletion, substitution and/or alteration of one ormore nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the siRNA or to one ormore internal nucleotides of the siRNA, or modifications that make thesiRNA resistant to nuclease digestion, or the substitution of one ormore nucleotides in the siRNA with deoxyribonucleotides.

One or both strands of the siRNA can also comprise a 3′ overhang. Asused herein, a “3′ overhang” refers to at least one unpaired nucleotideextending from the 3′-end of a duplexed RNA strand. Thus, in certainembodiments, the siRNA comprises at least one 3′ overhang of from 1 toabout 6 nucleotides (which includes ribonucleotides ordeoxyribonucleotides) in length, from 1 to about 5 nucleotides inlength, from 1 to about 4 nucleotides in length, or from about 2 toabout 4 nucleotides in length. In a particular embodiment, the 3′overhang is present on both strands of the siRNA, and is 2 nucleotidesin length. For example, each strand of the siRNA can comprise 3′overhangs of dithymidylic acid (“TT”) or diuridylic acid (“uu”).

The siRNA can be produced chemically or biologically, or can beexpressed from a recombinant plasmid or viral vector, as described abovefor the isolated miR gene products. Exemplary methods for producing andtesting dsRNA or siRNA molecules are described in U.S. Published PatentApplication No. 2002/0173478 to Gewirtz and in U.S. Published PatentApplication No. 2004/0018176 to Reich et al., the entire disclosures ofboth of which are incorporated herein by reference.

Expression of a given miR gene can also be inhibited by an antisensenucleic acid. As used herein, an “antisense nucleic acid” refers to anucleic acid molecule that binds to target RNA by means of RNA-RNA,RNA-DNA or RNA-peptide nucleic acid interactions, which alters theactivity of the target RNA. Antisense nucleic acids suitable for use inthe present methods are single-stranded nucleic acids (e.g., RNA, DNA,RNA-DNA chimeras, peptide nucleic acid (PNA)) that generally comprise anucleic acid sequence complementary to a contiguous nucleic acidsequence in a miR gene product. The antisense nucleic acid can comprisea nucleic acid sequence that is 50-100% complementary, 75-100%complementary, or 95-100% complementary to a contiguous nucleic acidsequence in a miR gene product.

Without wishing to be bound by any theory, it is believed that theantisense nucleic acids activate RNase H or another cellular nucleasethat digests the miR gene product/antisense nucleic acid duplex.

Antisense nucleic acids can also contain modifications to the nucleicacid backbone or to the sugar and base moieties (or their equivalent) toenhance target specificity, nuclease resistance, delivery or otherproperties related to efficacy of the molecule. Such modificationsinclude cholesterol moieties, duplex intercalators, such as acridine, orone or more nuclease-resistant groups.

Antisense nucleic acids can be produced chemically or biologically, orcan be expressed from a recombinant plasmid or viral vector, asdescribed above for the isolated miR gene products. Exemplary methodsfor producing and testing are within the skill in the art; see, e.g.,Stein and Cheng (1993), Science 261:1004 and U.S. Pat. No. 5,849,902 toWoolf et al., the entire disclosures of which are incorporated herein byreference.

Expression of a given miR gene can also be inhibited by an enzymaticnucleic acid. As used herein, an “enzymatic nucleic acid” refers to anucleic acid comprising a substrate binding region that hascomplementarity to a contiguous nucleic acid sequence of a miR geneproduct, and which is able to specifically cleave the miR gene product.The enzymatic nucleic acid substrate binding region can be, for example,50-100% complementary, 75-100% complementary, or 95-100% complementaryto a contiguous nucleic acid sequence in a miR gene product. Theenzymatic nucleic acids can also comprise modifications at the base,sugar, and/or phosphate groups.

Exemplary enzymatic nucleic acids for use in the present methods includede novo methyltransferases, including DNMT3A and DNMT3B, as described inthe Examples herein.

The enzymatic nucleic acids can be produced chemically or biologically,or can be expressed from a recombinant plasmid or viral vector, asdescribed above for the isolated miR gene products. Exemplary methodsfor producing and testing dsRNA or siRNA molecules are described inWerner and Uhlenbeck (1995), Nucl. Acids Res. 23:2092-96; Hammann et al.(1999), Antisense and Nucleic Acid Drug Dev. 9:25-31; and U.S. Pat. No.4,987,071 to Cech et al, the entire disclosures of which areincorporated herein by reference.

Administration of at least one miR gene product, or at least onecompound for inhibiting miR expression, will inhibit the proliferationof cancer cells in a subject who has a lung cancer.

As used herein, to “inhibit the proliferation of a cancer cell” means tokill the cell, or permanently or temporarily arrest or slow the growthof the cell Inhibition of cancer cell proliferation can be inferred ifthe number of such cells in the subject remains constant or decreasesafter administration of the miR gene products or miR geneexpression-inhibition compounds. An inhibition of cancer cellproliferation can also be inferred if the absolute number of such cellsincreases, but the rate of tumor growth decreases.

The number of cancer cells in the body of a subject can be determined bydirect measurement, or by estimation from the size of primary ormetastatic tumor masses. For example, the number of cancer cells in asubject can be measured by immunohistological methods, flow cytometry,or other techniques designed to detect characteristic surface markers ofcancer cells.

The size of a tumor mass can be ascertained by direct visualobservation, or by diagnostic imaging methods, such as X-ray, magneticresonance imaging, ultrasound, and scintigraphy. Diagnostic imagingmethods used to ascertain size of the tumor mass can be employed with orwithout contrast agents, as is known in the art. The size of a tumormass can also be ascertained by physical means, such as palpation of thetissue mass or measurement of the tissue mass with a measuringinstrument, such as a caliper.

The miR gene products or miR gene expression-inhibition compounds can beadministered to a subject by any means suitable for delivering thesecompounds to cancer cells of the subject. For example, the miR geneproducts or miR expression-inhibition compounds can be administered bymethods suitable to transfect cells of the subject with these compounds,or with nucleic acids comprising sequences encoding these compounds.

In one embodiment, the cells are transfected with a plasmid or viralvector comprising sequences encoding at least one miR gene product ormiR gene expression-inhibition compound.

Transfection methods for eukaryotic cells are well known in the art, andinclude, e.g., direct injection of the nucleic acid into the nucleus orpronucleus of a cell; electroporation; liposome transfer or transfermediated by lipophilic materials; receptor-mediated nucleic aciddelivery, bioballistic or particle acceleration; calcium phosphateprecipitation, and transfection mediated by viral vectors.

For example, cells can be transfected with a liposomal transfercompound, e.g., DOTAP(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methylsulfate,Boehringer-Mannheim) or an equivalent, such as LIPOFECTIN. The amount ofnucleic acid used is not critical to the practice of the invention;acceptable results may be achieved with 0.1-100 micrograms of nucleicacid/10⁵ cells. For example, a ratio of about 0.5 micrograms of plasmidvector in 3 micrograms of DOTAP per 10⁵ cells can be used.

A miR gene product or miR gene expression-inhibition compound can alsobe administered to a subject by any suitable enteral or parenteraladministration route. Suitable enteral administration routes for thepresent methods include, e.g., oral, rectal, or intranasal delivery.Suitable parenteral administration routes include, e.g., intravascularadministration (e.g., intravenous bolus injection, intravenous infusion,intra-arterial bolus injection, intra-arterial infusion and catheterinstillation into the vasculature); peri- and intra-tissue injection(e.g., peri-tumoral and intra-tumoral injection, intra-retinalinjection, or subretinal injection); subcutaneous injection ordeposition, including subcutaneous infusion (such as by osmotic pumps);direct application to the tissue of interest, for example by a catheteror other placement device (e.g., a retinal pellet or a suppository or animplant comprising a porous, non-porous, or gelatinous material); andinhalation. Particularly suitable administration routes are injection,infusion and direct injection into the tumor.

In the present methods, a miR gene product or miR gene productexpression-inhibition compound can be administered to the subject eitheras naked RNA, in combination with a delivery reagent, or as a nucleicacid (e.g., a recombinant plasmid or viral vector) comprising sequencesthat express the miR gene product or miR gene productexpression-inhibition compound. Suitable delivery reagents include,e.g., the Minis Transit TKO lipophilic reagent; lipofectin;lipofectamine; cellfectin; polycations (e.g., polylysine), andliposomes.

Recombinant plasmids and viral vectors comprising sequences that expressthe miR gene products or miR gene expression-inhibition compounds, andtechniques for delivering such plasmids and vectors to cancer cells, arediscussed herein and/or are well known in the art.

In a particular embodiment, liposomes are used to deliver a miR geneproduct or miR gene expression-inhibition compound (or nucleic acidscomprising sequences encoding them) to a subject. Liposomes can alsoincrease the blood half-life of the gene products or nucleic acids.Suitable liposomes for use in the invention can be formed from standardvesicle-forming lipids, which generally include neutral or negativelycharged phospholipids and a sterol, such as cholesterol. The selectionof lipids is generally guided by consideration of factors, such as thedesired liposome size and half-life of the liposomes in the bloodstream. A variety of methods are known for preparing liposomes, forexample, as described in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng.9:467; and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and5,019,369, the entire disclosures of which are incorporated herein byreference.

The liposomes for use in the present methods can comprise a ligandmolecule that targets the liposome to cancer cells. Ligands that bind toreceptors prevalent in cancer cells, such as monoclonal antibodies thatbind to tumor cell antigens, are preferred.

The liposomes for use in the present methods can also be modified so asto avoid clearance by the mononuclear macrophage system (“MMS”) andreticuloendothelial system (“RES”). Such modified liposomes haveopsonization-inhibition moieties on the surface or incorporated into theliposome structure. In a particularly preferred embodiment, a liposomeof the invention can comprise both an opsonization-inhibition moiety anda ligand.

Opsonization-inhibiting moieties for use in preparing the liposomes ofthe invention are typically large hydrophilic polymers that are bound tothe liposome membrane. As used herein, an opsonization-inhibiting moietyis “bound” to a liposome membrane when it is chemically or physicallyattached to the membrane, e.g., by the intercalation of a lipid-solubleanchor into the membrane itself, or by binding directly to active groupsof membrane lipids. These opsonization-inhibiting hydrophilic polymersform a protective surface layer that significantly decreases the uptakeof the liposomes by the MMS and RES; e.g., as described in U.S. Pat. No.4,920,016, the entire disclosure of which is incorporated herein byreference.

Opsonization-inhibiting moieties suitable for modifying liposomes arepreferably water-soluble polymers with a number-average molecular weightfrom about 500 to about 40,000 daltons, and more preferably from about2,000 to about 20,000 daltons. Such polymers include polyethylene glycol(PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG orPPG, and PEG or PPG stearate; synthetic polymers, such as polyacrylamideor poly N-vinyl pyrrolidone; linear, branched, or dendrimericpolyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcoholand polyxylitol to which carboxylic or amino groups are chemicallylinked, as well as gangliosides, such as ganglioside GM1. Copolymers ofPEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are alsosuitable. In addition, the opsonization-inhibiting polymer can be ablock copolymer of PEG and either a polyamino acid, polysaccharide,polyamidoamine, polyethyleneamine, or polynucleotide. Theopsonization-inhibiting polymers can also be natural polysaccharidescontaining amino acids or carboxylic acids, e.g., galacturonic acid,glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid,neuraminic acid, alginic acid, carrageenan; aminated polysaccharides oroligosaccharides (linear or branched); or carboxylated polysaccharidesor oligosaccharides, e.g., reacted with derivatives of carbonic acidswith resultant linking of carboxylic groups. Preferably, theopsonization-inhibiting moiety is a PEG, PPG, or a derivative thereof.Liposomes modified with PEG or PEG-derivatives are sometimes called“PEGylated liposomes.”

The opsonization-inhibiting moiety can be bound to the liposome membraneby any one of numerous well-known techniques. For example, anN-hydroxysuccinimide ester of PEG can be bound to aphosphatidyl-ethanolamine lipid-soluble anchor, and then bound to amembrane. Similarly, a dextran polymer can be derivatized with astearylamine lipid-soluble anchor via reductive amination usingNa(CN)BH₃ and a solvent mixture, such as tetrahydrofuran and water in a30:12 ratio at 60° C.

Liposomes modified with opsonization-inhibition moieties remain in thecirculation much longer than unmodified liposomes. For this reason, suchliposomes are sometimes called “stealth” liposomes. Stealth liposomesare known to accumulate in tissues fed by porous or “leaky”microvasculature. Thus, tissue characterized by such microvasculaturedefects, for example, lung tumors, will efficiently accumulate theseliposomes; see Gabizon, et al. (1988), Proc. Natl. Acad. Sci., U.S.A.,18:6949-53. In addition, the reduced uptake by the RES lowers thetoxicity of stealth liposomes by preventing significant accumulation ofthe liposomes in the liver and spleen. Thus, liposomes that are modifiedwith opsonization-inhibition moieties are particularly suited to deliverthe miR gene products or miR gene expression-inhibition compounds (ornucleic acids comprising sequences encoding them) to tumor cells.

The miR gene products or miR gene expression-inhibition compounds can beformulated as pharmaceutical compositions, sometimes called“medicaments,” prior to administering them to a subject, according totechniques known in the art. Accordingly, the invention encompassespharmaceutical compositions for treating a lung cancer.

In one embodiment, the pharmaceutical composition comprises at least oneisolated miR gene product, or an isolated variant or biologically-activefragment thereof, and a pharmaceutically-acceptable carrier. In aparticular embodiment, the at least one miR gene product corresponds toa miR gene product that has a decreased level of expression in lungcancer cells relative to suitable control cells. In certain embodimentsthe isolated miR gene product is selected from the group consisting ofmiR-29a, miR-29b, miR-29c, and combinations thereof.

In other embodiments, the pharmaceutical compositions of the inventioncomprise at least one miR expression-inhibition compound. In aparticular embodiment, the at least one miR gene expression-inhibitioncompound is specific for a miR gene whose expression is greater in lungcancer cells than control cells. In certain embodiments, the miR geneexpression-inhibition compound is specific for one or more miR geneproducts selected from the group consisting miR-29a, miR-29b, miR-29c,and combinations thereof.

Pharmaceutical compositions of the present invention are characterizedas being at least sterile and pyrogen-free. As used herein,“pharmaceutical compositions” include formulations for human andveterinary use. Methods for preparing pharmaceutical compositions of theinvention are within the skill in the art, for example as described inRemington's Pharmaceutical Science, 17th ed., Mack Publishing Company,Easton, Pa. (1985), the entire disclosure of which is incorporatedherein by reference.

The present pharmaceutical compositions comprise at least one miR geneproduct or miR gene expression-inhibition compound (or at least onenucleic acid comprising sequences encoding them) (e.g., 0.1 to 90% byweight), or a physiologically-acceptable salt thereof, mixed with apharmaceutically-acceptable carrier. In certain embodiments, thepharmaceutical compositions of the invention additionally comprise oneor more anti-cancer agents (e.g., chemotherapeutic agents). Thepharmaceutical formulations of the invention can also comprise at leastone miR gene product or miR gene expression-inhibition compound (or atleast one nucleic acid comprising sequences encoding them), which areencapsulated by liposomes and a pharmaceutically-acceptable carrier. Inone embodiment, the pharmaceutical composition comprises a miR gene orgene product that is one or more of miR-29a, miR-29b and miR-29c.

Especially suitable pharmaceutically-acceptable carriers are water,buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronicacid and the like.

In a particular embodiment, the pharmaceutical compositions of theinvention comprise at least one miR gene product or miR geneexpression-inhibition compound (or at least one nucleic acid comprisingsequences encoding them) that is resistant to degradation by nucleases.

One skilled in the art can readily synthesize nucleic acids that arenuclease resistant, for example, by incorporating one or moreribonucleotides that is modified at the 2′-position into the miR geneproduct. Suitable 2′-modified ribonucleotides include those modified atthe 2′-position with fluoro, amino, alkyl, alkoxy, and O-allyl.

Pharmaceutical compositions of the invention can also compriseconventional pharmaceutical excipients and/or additives. Suitablepharmaceutical excipients include stabilizers, antioxidants, osmolalityadjusting agents, buffers, and pH adjusting agents. Suitable additivesinclude, e.g., physiologically biocompatible buffers (e.g., tromethaminehydrochloride), additions of chelants (such as, for example, DTPA orDTPA-bisamide) or calcium chelate complexes (such as, for example,calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calciumor sodium salts (for example, calcium chloride, calcium ascorbate,calcium gluconate or calcium lactate). Pharmaceutical compositions ofthe invention can be packaged for use in liquid form, or can belyophilized.

For solid pharmaceutical compositions of the invention, conventionalnontoxic solid pharmaceutically-acceptable carriers can be used; forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharin, talcum, cellulose, glucose, sucrose,magnesium carbonate, and the like.

For example, a solid pharmaceutical composition for oral administrationcan comprise any of the carriers and excipients listed above and 10-95%,preferably 25%-75%, of the at least one miR gene product or miR geneexpression-inhibition compound (or at least one nucleic acid comprisingsequences encoding them). A pharmaceutical composition for aerosol(inhalational) administration can comprise 0.01-20% by weight,preferably 1%-10% by weight, of the at least one miR gene product or miRgene expression-inhibition compound (or at least one nucleic acidcomprising sequences encoding them) encapsulated in a liposome asdescribed above, and a propellant. A carrier can also be included asdesired; e.g., lecithin for intranasal delivery.

The pharmaceutical compositions of the invention can further compriseone or more anti-cancer agents. In a particular embodiment, thecompositions comprise at least one miR gene product or miR geneexpression-inhibition compound (or at least one nucleic acid comprisingsequences encoding them) and at least one chemotherapeutic agent.Chemotherapeutic agents that are suitable for the methods of theinvention include, but are not limited to, DNA-alkylating agents,anti-tumor antibiotic agents, anti-metabolic agents, tubulin stabilizingagents, tubulin destabilizing agents, hormone antagonist agents,topoisomerase inhibitors, protein kinase inhibitors, HMG-CoA inhibitors,CDK inhibitors, cyclin inhibitors, caspase inhibitors, metalloproteinaseinhibitors, antisense nucleic acids, triple-helix DNAs, nucleic acidsaptamers, and molecularly-modified viral, bacterial and exotoxic agents.Examples of suitable agents for the compositions of the presentinvention include, but are not limited to, cytidine arabinoside,methotrexate, vincristine, etoposide (VP-16), doxorubicin (adriamycin),cisplatin (CDDP), dexamethasone, arglabin, cyclophosphamide, sarcolysin,methylnitrosourea, fluorouracil, 5-fluorouracil (5FU), vinblastine,camptothecin, actinomycin-D, mitomycin C, hydrogen peroxide,oxaliplatin, irinotecan, topotecan, leucovorin, carmustine,streptozocin, CPT-11, taxol, tamoxifen, dacarbazine, rituximab,daunorubicin, 1-β-D-arabinofuranosylcytosine, imatinib, fludarabine,docetaxel, FOLFOX4.

There is also provided herein methods of identifying an inhibitor oftumorigenesis, comprising providing a test agent to a cell and measuringthe level of at least one miR gene product in the cell. In oneembodiment, the method comprises providing a test agent to a cell andmeasuring the level of at least one miR gene product associated withdecreased expression levels in cancer cells. An increase in the level ofthe miR gene product in the cell after the agent is provided, relativeto a suitable control cell (e.g., agent is not provided), is indicativeof the test agent being an inhibitor of tumorigenesis. In a particularembodiment, at least one miR gene product associated with decreasedexpression levels in cancer cells is selected from the group consistingof miR-29a, miR-29b, miR-29c, and combinations thereof.

In other embodiments, the method comprises providing a test agent to acell and measuring the level of at least one miR gene product associatedwith increased expression levels in cancer cells. A decrease in thelevel of the miR gene product in the cell after the agent is provided,relative to a suitable control cell (e.g., agent is not provided), isindicative of the test agent being an inhibitor of tumorigenesis. In aparticular embodiment, at least one miR gene product associated withincreased expression levels in cancer cells is selected from the groupconsisting of miR-29a, miR-29b, miR-29c, and combinations thereof.

Suitable agents include, but are not limited to drugs (e.g., smallmolecules, peptides), and biological macromolecules (e.g., proteins,nucleic acids). The agent can be produced recombinantly, synthetically,or it may be isolated (i.e., purified) from a natural source. Variousmethods for providing such agents to a cell (e.g., transfection) arewell known in the art, and several of such methods are describedhereinabove. Methods for detecting the expression of at least one miRgene product (e.g., Northern blotting, in situ hybridization, RT-PCR,expression profiling) are also well known in the art. Several of thesemethods are also described hereinabove.

The invention will now be illustrated by the following non-limitingexamples.

Example 1

miR-29s expression is inversely correlated to DNMT3A and 3B in lungcancer patients. In addition, miR-29s directly target both DNMT3A and3B. The enforced expression of miR-29s in lung cancer cell linesrestores normal patterns of DNA methylation, induces re-expression ofmethylation-silenced tumor suppressor genes (TSGs), such as FHIT, andWWOX and inhibits tumorigenicity both in vitro and in vivo.

These findings support a role of miR-29s in the epigenetic regulation ofNSCLC, providing a rationale for the development of miR-based strategiesfor the treatment of lung cancer.

172 matched non-neoplastic/primary NSCLC tissue pairs were analyzed byimmunohistochemical analysis of tissue microarrays (TMAs). As shown inFIG. 5, higher expression of DNMT3A protein was significantly associatedwith lower overall survival (P=0.029). Statistically significantcorrelations with survival were not observed for DNMT1 and DNTM3B inthis patient population.

To validate these miRNA-target interactions in vivo, the DNMT3A andDNTM3B complementary sites were cloned into the 3′UTR of the fireflyluciferase gene and co-transfected with miR-29a, mi-R29b or miR-29c inA459 (NSCLC) cells.

As shown in FIG. 2 a, all three miRNAs (miR-29a, mi-R29b or miR-29c)significantly reduced the luciferase activity with respect to thescrambled oligonucleotide. To assess whether ectopic expression ofindividual miR-29 sequences induces down-regulation of endogenous DNMT3Aand DNTM3B mRNA levels, we also performed quantitative RT-PCR (qRT-PCR)in A549 and H1299 lung cancer-derived cells, transfected with scrambledRNA or with miR-29s.

Overexpression of individual miR-29s induced marked reduction of DNMT3Aand DNMT3B mRNA levels (FIG. 2 b, upper), whereas silencing of miR-29swith antisense molecules, induced up-regulation of DNMT3A and DNMT3BmRNA levels (FIG. 2 b, lower) (results shown only for A549 cells).

To demonstrate that overexpression of miR-29s could downmodulate Dnmt3Aand 3B protein expression, we used a GFP-reporter vector, QBI-GFP25.

Briefly, we cloned the 3′UTRs of DNMT3A and DNMT3B downstream of the GFPencoding sequence of the QBI-GFP25 vector, allowing expression of afusion GFP protein containing the 3′UTR of DNMT3A or DNMT3B. A549 cellswere cotransfected with the GFP-3A/3B-3′UTR-vector plus miR-29a,miR-29b, miR-29c, or scrambled oligonucleotide. Marked reduction in GFPprotein expression was observed in cells transfected with miR-29s (FIG.2 c), especially GFP-3B-3′UTR protein; the protein expression resultswere consistent with those obtained by qRT-PCR, in which endogenousDNMT3B mRNA was more significantly reduced by expression of miR-29s(FIG. 2 b).

While not wishing to be bound by theory, it is now believed that thatthe preferential downregulation of DNMT3B compared to DNMT3A may bepossibly due to an overall higher number of predicted matching “seeds”of miR-29s with 3B 3′-UTR (3 for miR-29a, 1 for miR-29b, 1 for miR-29c)than with 3A 3′-UTR (1 for each miR-29).

In addition, the DNMT3B 3′UTR presents matching sites differing for nomore than 1 nucleotide for miR-29a and for miR-29b/c matches. Thus, thetransfection with any member of the miR-29 family may result in morerobust silencing of DNMT3B than DNMT3A, according to the “coordinateprinciple” that miRNAs may act cooperatively through multiple targetsites in one gene.

To show a direct, functional interaction of the DNMT3B 3′UTR withmiR-29b, a recently described detection method was used to detectmiRNA-mRNA complexes in eukaryotic cells by synthesizing cDNA on a mRNAtemplate using miRNAs as the endogenous cytoplasmic primer. Theendogenous miR-29b, at the Pictar-predicted site of interaction with 3′UTR, is able to function as a “natural” primer to initiate theretrotranscription of DNMT3B mRNA (FIG. 2 d).

It was then determined whether DNMT3A and DNMT3B mRNA expression isinversely correlated to the levels of miR-29s in primary NSCLC tissues.Fourteen (14) NSCLCs were analyzed for expression levels of DNMT3A andDNMT3B mRNAs and for miR-29a, miR-29b, and miR-29c expression byqRT-PCR. A statistically significant inverse correlation (FIG. 6) wasobserved between DNMT3A mRNA and miR-29a (P=0.02) and miR-29c (P=0.02).

A similar inverse correlation was observed for DNMT3B mRNA levels andmiR-29a (P=0.02) and miR-29c (P=0.04). Although there was a trend towardinverse correlation of DNMT3A and DNMT3B mRNA levels with miR-29b level,the association was not statistically significant (DNMT3A P=0.14, DNMT3BP=0.09), this may be due either to the small number of cancers analyzedor to the fact that while miR-29a and miR-29c are transcribed from onlyone chromosomal location, on chromosome 7 and 1 respectively, maturemiR-29b is transcribed from two different primary transcripts ondifferent chromosomes, the miR-29b-1/miR-29a cluster on 7q32.3 and themiR-29b-2/miR-29c cluster on 1q32.2. The probe used in qRT-PCR todetermine the mature product of miR-29b is unable to distinguish betweenthe miR-29b-1 or miR-29b-2 gene products.

The discovery that miR-29s target DNMT3A and DNMT3B shows thatexpression of these miRNAs contributes to the DNA epigeneticmodifications in cancer. To address this issue, A549 cells weretransfected with miR-29a, miR-29b, miR-29c or scrambled oligonucleotidesand analyzed global DNA methylation 48 and 72 h later, using an LC-MS/MSmethod.

As shown in FIG. 3 a, all three miR-29s reduced global DNA methylation,with respect to the control. The effect appeared more robust formiR-29b, with reduction of 30% after 48 h and 40% after 72 h. Thepercentage of global methylation reduction observed in cells treatedwith miR-29b is comparable to that observed with DNMT1 inhibitors suchas decitabine, and is partial with either approach. While not wishing tobe bound by theory, the inventor herein now believes that a more robustglobal DNA hypomethylation can be achieved combining decitabine (orother nucleoside analogs) with miR-29s thereby blocking both de novo andmaintenance DNMT pathways.

To characterize effects of the methylation changes on gene expression,the mRNA expression levels of two TSGs, FHIT and WWOX, which arefrequently silenced by promoter methylation in lung cancer wereanalyzed.

As shown in FIG. 3 b upper, 48 h after transfection of A549 cells, FHITexpression was increased by miR-29a, miR-29b and miR-29c expression by˜65%, 89%, and 74%, respectively, and the WWOX mRNA level was increasedby—40%, and 60% by miR-29a and 29b respectively; a similar trend wasobserved in H1299 cells (FIG. 3 b, lower).

Increased expression of both FHIT and WWOX proteins was also observed inboth cell lines (FIG. 3 c).

To determine if miR-29s regulate expression of FHIT and WWOX by alteringpromoter methylation of these genes, the methylation status of theregulatory region of FHIT and WWOX was examined using the MassARRAYsystem (quantitative high-throughput DNA methylation analysis) in A549and H1299 cells transfected with miR-29b. Two bisulfate reactions (onefor each gene CpG island) were designed, which covered 7 CpGs and 11CpGs for FHIT and WWOX respectively. In miR-29b transfected H1299 andA549 cells, the MassARRAY analysis for FHIT showed an average reductionof 19.1% and 54.3% methylation, respectively, whereas for WWOX in H1299showed an average reduction of 32.1% compared with the scrambledoligonucleotide (FIG. 3 d).

The effects of re-expression of miR-29s on tumorigenicity of A549 cellswere also assessed. The ectopic expression of miR-29s in A549 inhibitedin vitro cell growth (FIG. 4 a), and induced apoptosis with respect tothe scrambled control transfection (FIG. 4 b).

The inhibitory effect of miR-29s on A549 tumorigenicity was alsoobserved in vivo. Transfection with miR-29s inhibited the growth of A549engrafted tumors, with respect to mock and scrambled oligo transfectedcells (FIGS. 4 c, 4 d, 4 e), thus illustrating a likely antineoplasticeffect of these miRNAs.

Thus, this example shows that expression of miR-29 family members isinversely correlated with DNMT3A and DNMT3B expression in lung cancersand these miRNAs down-modulate expression levels of both enzymes.

Furthermore, enforced expression of these miRNAs in lung cancer cellsleads to reduced global DNA methylation, restores expression of TSGs andinhibits tumorigenicity both in vitro and in vivo. These results areuseful for developing novel epigenetic therapies using syntheticmiR-29s, alone or in combination with other treatments, to reactivatetumor suppressors and normalize aberrant patterns of methylation in lungcancer. Since loss of expression of miR-29 family members is observed inother common human malignancies, this approach may be extended to thetreatment of other human malignancies.

Methods.

Samples

We obtained 172 lung cancer samples, including squamous cell, adeno-,large cell and neuroendocrine large cell carcinomas, collectivelyreferred to as non small cell lung carcinomas (NSCLCs) from thePathology Core Facility at The Ohio State University to perform tissuemicroarrays (TMAs) for DNMTs expression. Clinical features (histologicaldiagnosis, sex, age, TNM status and survival time) were available forthese patients.

Primary lung cancer tissues (8 squamous carcinomas and 6adenocarcinomas) were purchased from the Cooperative Human TissueNetwork—Midwestern Division, Columbus, Ohio, to perform qRT-PCRanalysis. Total RNAs were isolated by TRIzol (Invitrogen, Carlsbad,Calif.) extraction, according to the manufacturer's instructions.

Tissue Microarrays

Tissue micro arrays (TMAs): each array contained 4 samples of each lungcancer along with multiple appropriate lung and other normal tissuespots. The TMAs, usually two for each antiserum, were stained withantisera against DNMT1, DNTM3A and DNMT3B proteins, and expression ofeach of these enzymes in lung cancer was compared with clinical featuresto seek significant correlations. DNMT1, DNMT3A and DNMT3B proteinexpression were assessed on the lung cancer TMAs, using DNMT1 antiserumfrom GeneTex (GTX13537, San Antonio, Tex.) at a dilution of 1:150;DNMT3A antiserum from Novus Biologicals (ab-4897, Littleton, Colo.) at adilution of 1:25 and DNTM3B antiserum from Abgent (AP1035a, San Diego,Calif.) at a dilution of 1:32. 4 micron sections from TMA blocks wereplaced on positively charged slides, placed in a 60° C. oven for 1 h,cooled, deparaffinized and rehydrated through xylene and graded ethanolsolutions to water. Slides were quenched for 5 min in 3% hydrogenperoxide to block endogenous peroxidase. Antigens were retrieved in TRS(Dako, Carpinteria, Calif.) solution at 95 C, 25 min Slides were exposedto primary antisera for 1 h at room temperature and to secondaryantisera (1:200) for 20 min, room temperature; secondary antisera weregoat anti-mouse for DNMT1 and goat anti-rabbit for DNMT3A and DMT3B. Allslides were blocked for endogenous biotin prior to application of thebiotinylated secondary antisera. Chromogen detection was with VectastainElite (Vector, cat# PK-6100) for 30 min The substrate chromogen wasDAB+(Dako, cat# K3468). Slides were counterstained with hematoxylin,dehydrated through graded ethanol solutions and cover-slipped.

TMAs were read and scored by a pathologist who was blinded to clinicalfeatures; expression scores were determined by multiplying the percentof positive cells in an individual sample by the intensity of staining;the intensity of staining was assessed on a scale from 1 to 3, where 1was the least intense staining and 3 was the most intense. For example,a sample with 10% positive cells with intensity 3 was assigned a scoreof 30, the same score as a sample with 30% positive cells with intensity1.

Quantitative RT-PCR.

Quantitative RT-PCR (qRT-PCR) analysis for miRNAs was performed intriplicate with the TaqMan MicroRNA assays kit (Applied Biosystems,Foster City, Calif.), according to the manufacturer's instructions. 18SRNA was used for normalization; qRT-PCR analyses for other genes ofinterest were performed as previously described¹. RNA was reversetranscribed to cDNA with gene-specific primers and IQ SYBR GreenSupermix (Biorad, Hercules, Calif.). GAPDH served as normalizationcontrol. For the silencing of miR-29s, A549 and H1299 cells weretransfected in E-well plates by using Lipofectamine 2000 reagent(Invitrogen), according to the manufacturer's protocol, with 100 nM(final) of antisense miR-29a, 29b-1, 29c or scrambled antisense miR(Fidelity Systems, Gaithersburg, Md.).

Cell Culture.

A549 and H1299 lung cancer cells from the American Type CultureCollection (Manassas, Va.) were maintained in RPMI medium 1640 with 10%FBS and antibiotics (100 U/ml penicillin, and 100 ng/ml streptomycin).

Luciferase Reporter Assay for Targeting DNMT 3′UTRs.

For luciferase reporter experiments a DNMT3A 3′UTR segment of 979 bp anda DNMT3B 3′UTR segment of 978 bp were amplified by PCR from humangenomic DNA and inserted into the pGL3-control vector with SV40 promoter(Promega), using the XbaI site immediately upstream from the stop codonof luciferase. The following sets of primers were used to generatespecific fragments:

DNMT3A-UTR Fw: [SEQ ID NO: 15] 5′-GCTCTAGAGCCGAAAAGGGTTGGACATCAT-3′,DNMT3A-UTR Rv: [SEQ ID NO: 16] 5′-GCTCTAGAGCGCCGAGGGAGTCTCCTTTTA-3′;DNMT3B-UTR Fw: [SEQ ID NO: 17] 5′-GCTCTAGAGCTAGGTAGCAACGTGGCTTTT-3′,DNMT3B-UTR Rv: [SEQ ID NO: 18] 5′-GCTCTAGAGCGCCCCACAAAACTTGTCAAC-3′.

The amplified 3′UTR of DNMT3A contains an XbaI restriction site inposition 583, so we cloned separately the upstream 3′UTR (DNMT3A3′-UTRup=583 bp) and the downstream fragment (DNMT3A 3′-UTRdown=396 bp)into the pGL3 vectors. The predicted match seed of miR-29s is located inthe DNMT3A 3′-UTR down fragment, which was used to perform theluciferase assay.

A549 cells were co-transfected in 12-well plates by using Lipofectamine2000 reagent (Invitrogen), according to the manufacturer's protocol,with 0.4 μg of the firefly luciferase report vector and 0.08 μg of thecontrol vector containing Renilla luciferase pRL-TK vector (Promega).For each well, 100 nM (final) of precursor miR-29a, miR-29b-1, miR-29cor scrambled miR (Ambion) was used. Firefly and Renilla luciferaseactivities were measured consecutively by using dual-luciferase assays(Promega), 24 h after the transfection. The experiments were performedin triplicate.

GFP-Repression Constructs to Assess Effect of DNMT 3′UTRs on ProteinExpression.

For GFP-repression, a DNMT3A 3′UTR segment of 1472 bp and a DNMT3B 3′UTRsegment of 1566 bp (corresponding to the whole length of the 3′UTRs)were amplified by PCR from human genomic DNA and inserted into theQBI-GFP25 vector (Autofluorescent Proteins, Canada), using theBamHI-EcoRI cloning sites located 3′ of the GFP encoding sequence of thevector (which has no stop codon at the end of the GFP coding sequence).The following primer sets were used to generate specific fragments:

DNMT3A-GFP Fw: [SEQ ID NO: 19] 5′-CGGGATCCGCAGGATAGCCAAGTTCAGC-3′,DNMT3A-GFP Rv: [SEQ ID NO: 20] 5′-CCCAAGCTTAAGTGAGAAACTGGGCCTGA-3′;DNMT3B-GFP Fw: [SEQ ID NO: 21] 5′-CGGGATCCCTCGATCAAACAGGGGAAAA-3′,DNMT3B-GFP Rv: [SEQ ID NO: 22] 5′-CCCAAGCTTGTTACGTCGTGGCTCCAGTT-3′.

A549 cells were co-transfected in 12-well plates using Lipofectamine2000 reagent (Invitrogen) according to the manufacturer's protocol with2 μg of the GFP repression vector containing the 3′UTR of DNMT3A(QBI-GFP25-DNMT3A) or the 3′UTR of DNMT3B (QBI-GFP25-DNMT3B) and with100 nM (final) of precursor miR-29a, miR-29b-1, miR-29c, or scrambledoligonucleotide (Ambion). As an additional control, a group of cells wasalso transfected with the GFP vector (no miR). Cells were harvestedafter 24 h. Protein extraction and immunoblot analysis were performed aspreviously described. The following primary antisera were used: rabbitpolyclonal anti-GFP, 1:1000 (Novus Biologicals, Littleton, Colo.).

Detection of miR29b-DNMT3B RNA Complexes.

To detect miR-29b-DNMT3B RNA complexes, we used the method described byVatolin S. et al. to determine if endogenous miR-29b was able to serveas primer for retrotranscription of DNMT3B mRNA in A549 cells. The cDNAswere cloned in pCR2.1-TOPO Vector (Invitrogen). The following sets ofprimers and adapter sequence were used (GSP meaning gene specificprimer):

GSP-DNMT3B: [SEQ ID NO: 23] 5′-GAGATGACAGGGAAAACTGC-3′; GSP-DNMT3B 5N:[SEQ ID NO: 24] 5′-ACAGGGAAAACTGCAAAGCT-3′; Adapter: [SEQ ID NO: 25]5′-CGACTGGAGCACGAGGACACTGACATGGACTGAAGGAGTAGA AA-3′; Adapter 5N:[SEQ ID NO: 26] 5′-CTGAAGGAGTAGAAA-3′.

Primers 5N represent nested primers from the adapter and GSP sequenceused to sensitize the detection of the PCR bands.

Global Methylation Studies.

The global methylation status of A549 cells after transfection withscrambled miRNA and with miR-29s, was determined as previouslydescribed. For this assay, 2×10⁶ A549 cells were transfected asdescribed above for the luciferase assay, and collected 48 and 72 hlater.

Quantitative DNA Methylation.

Quantitative DNA methylation analysis of the regulatory regions of FHITand WWOX was done using the EpiTYPER methylation analysis assay(Sequenom, San Diego, Calif.). Two bisulfite reactions (one for eachgene CpG island) were designed, which covered 7 CpGs and 11 CpGs forFHIT and WWOX respectively. The DNA of scrambled-, ormiR-29b-transfected A549/H1299 was extracted 48 h after the transfectionand 1 μg of DNA was bisulfite treated, in vitro transcribed, cleaved byRnase A, and subjected to matrix-assisted laser desorptionionization-time of flight (MALDI-TOF) mass spectrometry analysis todetermine methylation patterns, as described. The following primers wereused to amplify the regulatory regions of the FHIT and WWOX genes:

FHIT Fw: [SEQ ID NO: 27] 5′-GGGGAGGTAAGTTTAAGTGGAATATTGTT-3′ FHIT Rv:[SEQ ID NO: 28] 5′-CACCCCCAAAACCAAAAACTATAAC-3′ WWOX Fw: [SEQ ID NO: 29]5′-TTGAAAGAAAGTTTTTTAAAATTAGGAAAT-3′ WWOX Rv: [SEQ ID NO: 30]5′-TCAAAAAAACAAAACCTAAAAAAAA-3′.

The heat map in FIG. 3 d was created using Heatmap builder version 1.0by Stanford University.

Western Blot Analysis for the FHIT and WWOX Proteins.

Protein extraction and immunoblot analysis were performed as previouslydescribed. The following primary antisera were used: rabbit polyclonalanti-FHIT, 1:1000 (Zymed, San Francisco, Calif.); mouse monoclonalanti-WWOX, 1:500. Quantitation of the signal for FHIT, WWOX and Gapdhwas performed by using a Molecular Dynamics Personal Densitometer SI andIMAGEQUANT 5.2 software (Image Products International, Chantilly, Va.).

Cell Growth Curve.

A549 cells (5×10⁴) were plated in 6× multi-well plates and transfected,after 24 hours, with scrambled oligonucleotides or miR-29soligonucleotides from Ambion at a final concentration of 100 nM, withLipofectamine 2000 (Invitrogen), according to manufacturer's protocol.As a control also not transfected (mock) cells were included. Cells wereharvested and counted at 24 h intervals using a ViCell counter (BeckmanCoulter, Fullerton, Calif.). Each sample was run in triplicate.

Apoptosis and Flow Cytometric Studies.

A549 cells (2×10⁵) were transfected with scrambled oligonucleotides ormiR-29s oligonucleotides from Ambion at a final concentration of 100 nM,with Lipofectamine 2000 (Invitrogen), according to manufacturer'sprotocol. After 24 h cells were resuspended in binding buffer containingannexin V-fluorescein isothiocyanate (FITC) and propidium iodideaccording to the supplier's instructions (BD Biosciences, San Diego,Calif.), and assessed by flow cytometry using a Beckman-Coulter modelEPICS XL cytometer (Beckman-Coulter). Each sample was run in triplicate.

In Vivo Studies.

Animal studies were performed according to institutional guidelines.A549 cells were transfected in vitro with 100 nM (final concentration)of scrambled (Scr) oligonucleotides, or miR-29a, miR-29b, or miR-29c, orwere mock-transfected by using Lipofectamine 2000 reagent (Invitrogen),according to the manufacturer's protocol. At 48 h after transfection,3×10⁶ viable cells were injected subcutaneously into the left flanks of6-wk-old female nude mice (Charles River Breeding Laboratories,Wilmington, Mass.), five mice per group. Tumor diameters were measuredafter 7 days from injection and then every 5 days. At 21 days afterinjection, mice were sacrificed and tumors were weighted after necropsy.Tumor volumes were determined by using the equation V (in mm³)=A×B²/2,where A is the largest diameter and B is the perpendicular diameter.

Statistical Analysis. P values were two-sided and obtained using theSPSS software package (SPSS 10.0).

Overall survival was calculated from the time of diagnosis until thedate of last follow-up. Data were censored for patients who were aliveat the time of last follow-up. To perform the survival analysis andgenerate a Kaplan-Meier (KM) plot, DNMT1, DNMT3A and DNMT3B levelsmeasured by immunohistochemical staining, were converted into discretevariables by splitting the samples into two classes (high and lowexpression, according to the DNMT score <10 (low) or >10 (high)).Survival curves were obtained for each group and compared by using thelog-rank test. To assess correlation between miRNA expression and DNMTexpression we used Pearson correlation and linear regression analysis(SPSS package). These functions examine each pair of measurements (onefrom the miRNA and the other from DNMTs) to determine if the twovariables tend to move together or in the opposite direction, that is ifthe larger values from the miRNA (high expression) are associated withthe lower values from DNMT expression.

Example 2 Methods, Reagents and Kits for Diagnosing, Staging,Prognosing, Monitoring and Treating Lung Cancer-Related Diseases

It is to be understood that all examples herein are to be considerednon-limiting in their scope. Various aspects are described in furtherdetail in the following subsections.

Diagnostic Methods

In one embodiment, there is provided a diagnostic method of assessingwhether a patient has a lung cancer-related disease or has higher thannormal risk for developing a lung cancer-related disease, comprising thesteps of comparing the level of expression of a marker in a patientsample and the normal level of expression of the marker in a control,e.g., a sample from a patient without a lung cancer-related disease.

A significantly higher level of expression of the marker in the patientsample as compared to the normal level is an indication that the patientis afflicted with a lung cancer-related disease or has higher thannormal risk for developing a lung cancer-related disease.

The markers are selected such that the positive predictive value of themethods is at least about 10%, and in certain non-limiting embodiments,about 25%, about 50% or about 90%. Also preferred for use in the methodsare markers that are differentially expressed, as compared to normalcells, by at least two-fold in at least about 20%, and in certainnon-limiting embodiments, about 50% or about 75%.

In one diagnostic method of assessing whether a patient is afflictedwith a lung cancer-related disease (e.g., new detection (“screening”),detection of recurrence, reflex testing), the method comprisescomparing: a) the level of expression of a marker in a patient sample,and b) the normal level of expression of the marker in a controlnon-lung cancer-related disease sample. A significantly higher level ofexpression of the marker in the patient sample as compared to the normallevel is an indication that the patient is afflicted with a lungcancer-related disease.

There is also provided diagnostic methods for assessing the efficacy ofa therapy for inhibiting a lung cancer-related disease in a patient.Such methods comprise comparing: a) expression of a marker in a firstsample obtained from the patient prior to providing at least a portionof the therapy to the patient, and b) expression of the marker in asecond sample obtained from the patient following provision of theportion of the therapy. A significantly lower level of expression of themarker in the second sample relative to that in the first sample is anindication that the therapy is efficacious for inhibiting a lungcancer-related disease in the patient.

It will be appreciated that in these methods the “therapy” may be anytherapy for treating a lung cancer-related disease including, but notlimited to, pharmaceutical compositions, gene therapy and biologictherapy such as the administering of antibodies and chemokines. Thus,the methods described herein may be used to evaluate a patient before,during and after therapy, for example, to evaluate the reduction indisease state.

In certain aspects, the diagnostic methods are directed to therapy usinga chemical or biologic agent. These methods comprise comparing: a)expression of a marker in a first sample obtained from the patient andmaintained in the presence of the chemical or biologic agent, and b)expression of the marker in a second sample obtained from the patientand maintained in the absence of the agent. A significantly lower levelof expression of the marker in the second sample relative to that in thefirst sample is an indication that the agent is efficacious forinhibiting a lung cancer-related disease in the patient. In oneembodiment, the first and second samples can be portions of a singlesample obtained from the patient or portions of pooled samples obtainedfrom the patient.

Methods for Assessing Prognosis

There is also provided a monitoring method for assessing the progressionof a lung cancer-related disease in a patient, the method comprising: a)detecting in a patient sample at a first time point, the expression of amarker; b) repeating step a) at a subsequent time point in time; and c)comparing the level of expression detected in steps a) and b), andtherefrom monitoring the progression of a lung cancer-related disease inthe patient. A significantly higher level of expression of the marker inthe sample at the subsequent time point from that of the sample at thefirst time point is an indication that the lung cancer-related diseasehas progressed, whereas a significantly lower level of expression is anindication that the lung cancer-related disease has regressed.

There is further provided a diagnostic method for determining whether alung cancer-related disease has worsened or is likely to worsen in thefuture, the method comprising comparing: a) the level of expression of amarker in a patient sample, and b) the normal level of expression of themarker in a control sample. A significantly higher level of expressionin the patient sample as compared to the normal level is an indicationthat the lung cancer-related disease has worsened or is likely to worsenin the future.

Methods for Assessing Inhibitory, Therapeutic and/or HarmfulCompositions

There is also provided a test method for selecting a composition forinhibiting a lung cancer-related disease in a patient. This methodcomprises the steps of: a) obtaining a sample comprising cells from thepatient; b) separately maintaining aliquots of the sample in thepresence of a plurality of test compositions; c) comparing expression ofa marker in each of the aliquots; and d) selecting one of the testcompositions which significantly reduces the level of expression of themarker in the aliquot containing that test composition, relative to thelevels of expression of the marker in the presence of the other testcompositions.

There is additionally provided a test method of assessing the harmfulpotential of a compound in causing a lung cancer-related disease. Thismethod comprises the steps of: a) maintaining separate aliquots of cellsin the presence and absence of the compound; and b) comparing expressionof a marker in each of the aliquots. A significantly higher level ofexpression of the marker in the aliquot maintained in the presence ofthe compound, relative to that of the aliquot maintained in the absenceof the compound, is an indication that the compound possesses suchharmful potential.

In addition, there is further provided a method of inhibiting a lungcancer-related disease in a patient. This method comprises the steps of:a) obtaining a sample comprising cells from the patient; b) separatelymaintaining aliquots of the sample in the presence of a plurality ofcompositions; c) comparing expression of a marker in each of thealiquots; and d) administering to the patient at least one of thecompositions which significantly lowers the level of expression of themarker in the aliquot containing that composition, relative to thelevels of expression of the marker in the presence of the othercompositions.

The level of expression of a marker in a sample can be assessed, forexample, by detecting the presence in the sample of: the correspondingmarker protein or a fragment of the protein (e.g. by using a reagent,such as an antibody, an antibody derivative, an antibody fragment orsingle-chain antibody, which binds specifically with the protein orprotein fragment) the corresponding marker nucleic acid (e.g. anucleotide transcript, or a complement thereof), or a fragment of thenucleic acid (e.g. by contacting transcribed polynucleotides obtainedfrom the sample with a substrate having affixed thereto one or morenucleic acids having the entire or a segment of the nucleic acidsequence or a complement thereof) a metabolite which is produceddirectly (i.e., catalyzed) or indirectly by the corresponding markerprotein.

Any of the aforementioned methods may be performed using at least one ora plurality (e.g., 2, 3, 5, or 10 or more) of lung cancer-relateddisease markers. In such methods, the level of expression in the sampleof each of a plurality of markers, at least one of which is a marker, iscompared with the normal level of expression of each of the plurality ofmarkers in samples of the same type obtained from control humans notafflicted with a lung cancer-related disease. A significantly altered(i.e., increased or decreased as specified in the above-describedmethods using a single marker) level of expression in the sample of oneor more markers, or some combination thereof, relative to that marker'scorresponding normal or control level, is an indication that the patientis afflicted with a lung cancer-related disease. For all of theaforementioned methods, the marker(s) are selected such that thepositive predictive value of the method is at least about 10%.

Examples of Candidate Agents

The candidate agents may be pharmacologic agents already known in theart or may be agents previously unknown to have any pharmacologicalactivity. The agents may be naturally arising or designed in thelaboratory. They may be isolated from microorganisms, animals or plants,or may be produced recombinantly, or synthesized by any suitablechemical method. They may be small molecules, nucleic acids, proteins,peptides or peptidomimetics. In certain embodiments, candidate agentsare small organic compounds having a molecular weight of more than 50and less than about 2,500 daltons. Candidate agents comprise functionalgroups necessary for structural interaction with proteins. Candidateagents are also found among biomolecules including, but not limited to:peptides, saccharides, fatty acids, steroids, purines, pyrimidines,derivatives, structural analogs or combinations thereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. There are, for example,numerous means available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. In certain embodiments, thecandidate agents can be obtained using any of the numerous approaches incombinatorial library methods art, including, by non-limiting example:biological libraries; spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the “one-bead one-compound” library method; and syntheticlibrary methods using affinity chromatography selection.

In certain further embodiments, certain pharmacological agents may besubjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

The same methods for identifying therapeutic agents for treating a lungcancer-related disease can also be used to validate leadcompounds/agents generated from in vitro studies.

The candidate agent may be an agent that up- or down-regulates one ormore lung cancer-related disease response pathways. In certainembodiments, the candidate agent may be an antagonist that affects suchpathway.

Methods for Treating a Lung Cancer-Related Disease

There is provided herein methods for treating, inhibiting, relieving orreversing a lung cancer-related disease response. In the methodsdescribed herein, an agent that interferes with a signaling cascade isadministered to an individual in need thereof, such as, but not limitedto, lung cancer-related disease patients in whom such complications arenot yet evident and those who already have at least one lungcancer-related disease response.

In the former instance, such treatment is useful to prevent theoccurrence of such lung cancer-related disease response and/or reducethe extent to which they occur. In the latter instance, such treatmentis useful to reduce the extent to which such lung cancer-related diseaseresponse occurs, prevent their further development or reverse the lungcancer-related disease response.

In certain embodiments, the agent that interferes with the lungcancer-related disease response cascade may be an antibody specific forsuch response.

Expression of a Marker

Expression of a marker can be inhibited in a number of ways, including,by way of a non-limiting example, an antisense oligonucleotide can beprovided to the lung cancer-related disease cells in order to inhibittranscription, translation, or both, of the marker(s). Alternately, apolynucleotide encoding an antibody, an antibody derivative, or anantibody fragment which specifically binds a marker protein, andoperably linked with an appropriate promoter/regulator region, can beprovided to the cell in order to generate intracellular antibodies whichwill inhibit the function or activity of the protein. The expressionand/or function of a marker may also be inhibited by treating the lungcancer-related disease cell with an antibody, antibody derivative orantibody fragment that specifically binds a marker protein. Using themethods described herein, a variety of molecules, particularly includingmolecules sufficiently small that they are able to cross the cellmembrane, can be screened in order to identify molecules which inhibitexpression of a marker or inhibit the function of a marker protein. Thecompound so identified can be provided to the patient in order toinhibit lung cancer-related disease cells of the patient.

Any marker or combination of markers, as well as any certain markers incombination with the markers, may be used in the compositions, kits andmethods described herein. In general, it is desirable to use markers forwhich the difference between the level of expression of the marker inlung cancer-related disease cells and the level of expression of thesame marker in normal lung cells is as great as possible. Although thisdifference can be as small as the limit of detection of the method forassessing expression of the marker, it is desirable that the differencebe at least greater than the standard error of the assessment method,and, in certain embodiments, a difference of at least 2-, 3-, 4-, 5-,6-, 7-, 8-, 9-, 10-, 15-, 20-, 100-, 500-, 1000-fold or greater than thelevel of expression of the same marker in normal tissue.

It is recognized that certain marker proteins are secreted to theextracellular space surrounding the cells. These markers are used incertain embodiments of the compositions, kits and methods, owing to thefact that such marker proteins can be detected in a lungcancer-associated body fluid sample, which may be more easily collectedfrom a human patient than a tissue biopsy sample. In addition, in vivotechniques for detection of a marker protein include introducing into asubject a labeled antibody directed against the protein. For example,the antibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

In order to determine whether any particular marker protein is asecreted protein, the marker protein is expressed in, for example, amammalian cell, such as a human lung line, extracellular fluid iscollected, and the presence or absence of the protein in theextracellular fluid is assessed (e.g. using a labeled antibody whichbinds specifically with the protein).

It will be appreciated that patient samples containing lung cells may beused in the methods described herein. In these embodiments, the level ofexpression of the marker can be assessed by assessing the amount (e.g.,absolute amount or concentration) of the marker in a sample. The cellsample can, of course, be subjected to a variety of post-collectionpreparative and storage techniques (e.g., nucleic acid and/or proteinextraction, fixation, storage, freezing, ultrafiltration, concentration,evaporation, centrifugation, etc.) prior to assessing the amount of themarker in the sample.

It will also be appreciated that the markers may be shed from the cellsinto the digestive system, the blood stream and/or interstitial spaces.The shed markers can be tested, for example, by examining the serum orplasma.

The compositions, kits and methods can be used to detect expression ofmarker proteins having at least one portion which is displayed on thesurface of cells which express it. For example, immunological methodsmay be used to detect such proteins on whole cells, or computer-basedsequence analysis methods may be used to predict the presence of atleast one extracellular domain (i.e., including both secreted proteinsand proteins having at least one cell-surface domain) Expression of amarker protein having at least one portion which is displayed on thesurface of a cell which expresses it may be detected without necessarilylysing the cell (e.g., using a labeled antibody which binds specificallywith a cell-surface domain of the protein).

Expression of a marker may be assessed by any of a wide variety ofmethods for detecting expression of a transcribed nucleic acid orprotein. Non-limiting examples of such methods include immunologicalmethods for detection of secreted, cell-surface, cytoplasmic or nuclearproteins, protein purification methods, protein function or activityassays, nucleic acid hybridization methods, nucleic acid reversetranscription methods and nucleic acid amplification methods.

In a particular embodiment, expression of a marker is assessed using anantibody (e.g., a radio-labeled, chromophore-labeled,fluorophore-labeled or enzyme-labeled antibody), an antibody derivative(e.g., an antibody conjugated with a substrate or with the protein orligand of a protein-ligand pair), or an antibody fragment (e.g., asingle-chain antibody, an isolated antibody hypervariable domain, etc.)which binds specifically with a marker protein or fragment thereof,including a marker protein which has undergone all or a portion of itsnormal post-translational modification.

In another particular embodiment, expression of a marker is assessed bypreparing mRNA/cDNA (i.e., a transcribed polynucleotide) from cells in apatient sample, and by hybridizing the mRNA/cDNA with a referencepolynucleotide which is a complement of a marker nucleic acid, or afragment thereof. cDNA can, optionally, be amplified using any of avariety of polymerase chain reaction methods prior to hybridization withthe reference polynucleotide; preferably, it is not amplified.Expression of one or more markers can likewise be detected usingquantitative PCR to assess the level of expression of the marker(s).Alternatively, any of the many methods of detecting mutations orvariants (e.g., single nucleotide polymorphisms, deletions, etc.) of amarker may be used to detect occurrence of a marker in a patient.

In a related embodiment, a mixture of transcribed polynucleotidesobtained from the sample is contacted with a substrate having fixedthereto a polynucleotide complementary to or homologous with at least aportion (e.g., at least 7, 10, 15, 20, 25, 30, 40, 50, 100, 500, or morenucleotide residues) of a marker nucleic acid. If polynucleotidescomplementary to or homologous with are differentially detectable on thesubstrate (e.g., detectable using different chromophores orfluorophores, or fixed to different selected positions), then the levelsof expression of a plurality of markers can be assessed simultaneouslyusing a single substrate (e.g., a “gene chip” microarray ofpolynucleotides fixed at selected positions). When a method of assessingmarker expression is used which involves hybridization of one nucleicacid with another, it is desired that the hybridization be performedunder stringent hybridization conditions.

In certain embodiments, the biomarker assays can be performed using massspectrometry or surface plasmon resonance. In various embodiment, themethod of identifying an agent active against a lung cancer-relateddisease can include a) providing a sample of cells containing one ormore markers or derivative thereof; b) preparing an extract from saidcells; c) mixing said extract with a labeled nucleic acid probecontaining a marker binding site; and, d) determining the formation of acomplex between the marker and the nucleic acid probe in the presence orabsence of the test agent. The determining step can include subjectingsaid extract/nucleic acid probe mixture to an electrophoretic mobilityshift assay.

In certain embodiments, the determining step comprises an assay selectedfrom an enzyme linked immunoabsorption assay (ELISA), fluorescence basedassays and ultra high throughput assays, for example surface plasmonresonance (SPR) or fluorescence correlation spectroscopy (FCS) assays.In such embodiments, the SPR sensor is useful for direct real-timeobservation of biomolecular interactions since SPR is sensitive tominute refractive index changes at a metal-dielectric surface. SPR is asurface technique that is sensitive to changes of 10⁵ to 10⁻⁶ refractiveindex (R1) units within approximately 200 nm of the SPR sensor/sampleinterface. Thus, SPR spectroscopy is useful for monitoring the growth ofthin organic films deposited on the sensing layer.

Because the compositions, kits, and methods rely on detection of adifference in expression levels of one or more markers, it is desiredthat the level of expression of the marker is significantly greater thanthe minimum detection limit of the method used to assess expression inat least one of normal cells and lung cancer-affected cells.

It is understood that by routine screening of additional patient samplesusing one or more of the markers, it will be realized that certain ofthe markers are over-expressed in cells of various types, includingspecific lung cancer-related diseases.

In addition, as a greater number of patient samples are assessed forexpression of the markers and the outcomes of the individual patientsfrom whom the samples were obtained are correlated, it will also beconfirmed that altered expression of certain of the markers are stronglycorrelated with a lung cancer-related disease and that alteredexpression of other markers are strongly correlated with other diseases.The compositions, kits, and methods are thus useful for characterizingone or more of the stage, grade, histological type, and nature of a lungcancer-related disease in patients.

When the compositions, kits, and methods are used for characterizing oneor more of the stage, grade, histological type, and nature of a lungcancer-related disease in a patient, it is desired that the marker orpanel of markers is selected such that a positive result is obtained inat least about 20%, and in certain embodiments, at least about 40%, 60%,or 80%, and in substantially all patients afflicted with a lungcancer-related disease of the corresponding stage, grade, histologicaltype, or nature. The marker or panel of markers invention can beselected such that a positive predictive value of greater than about 10%is obtained for the general population (in a non-limiting example,coupled with an assay specificity greater than 80%).

When a plurality of markers are used in the compositions, kits, andmethods, the level of expression of each marker in a patient sample canbe compared with the normal level of expression of each of the pluralityof markers in non-lung cancer samples of the same type, either in asingle reaction mixture (i.e. using reagents, such as differentfluorescent probes, for each marker) or in individual reaction mixturescorresponding to one or more of the markers. In one embodiment, asignificantly increased level of expression of more than one of theplurality of markers in the sample, relative to the corresponding normallevels, is an indication that the patient is afflicted with a lungcancer-related disease. When a plurality of markers is used, 2, 3, 4, 5,8, 10, 12, 15, 20, 30, or 50 or more individual markers can be used; incertain embodiments, the use of fewer markers may be desired.

In order to maximize the sensitivity of the compositions, kits, andmethods (i.e. by interference attributable to cells of non-lung originin a patient sample), it is desirable that the marker used therein be amarker which has a restricted tissue distribution, e.g., normally notexpressed in a non-lung tissue.

It is recognized that the compositions, kits, and methods will be ofparticular utility to patients having an enhanced risk of developing alung cancer-related disease and their medical advisors. Patientsrecognized as having an enhanced risk of developing a lungcancer-related disease include, for example, patients having a familialhistory of a lung cancer-related disease.

The level of expression of a marker in normal human lung tissue can beassessed in a variety of ways. In one embodiment, this normal level ofexpression is assessed by assessing the level of expression of themarker in a portion of lung cells which appear to be normal and bycomparing this normal level of expression with the level of expressionin a portion of the lung cells which is suspected of being abnormal.Alternately, and particularly as further information becomes availableas a result of routine performance of the methods described herein,population-average values for normal expression of the markers may beused. In other embodiments, the ‘normal’ level of expression of a markermay be determined by assessing expression of the marker in a patientsample obtained from a non-lung cancer-afflicted patient, from a patientsample obtained from a patient before the suspected onset of a lungcancer-related disease in the patient, from archived patient samples,and the like.

There is also provided herein compositions, kits, and methods forassessing the presence of lung cancer-related disease cells in a sample(e.g. an archived tissue sample or a sample obtained from a patient).These compositions, kits, and methods are substantially the same asthose described above, except that, where necessary, the compositions,kits, and methods are adapted for use with samples other than patientsamples. For example, when the sample to be used is a parafinized,archived human tissue sample, it can be necessary to adjust the ratio ofcompounds in the compositions, in the kits, or the methods used toassess levels of marker expression in the sample.

Methods of Producing Antibodies

There is also provided herein a method of making an isolated hybridomawhich produces an antibody useful for assessing whether a patient isafflicted with a lung cancer-related disease. In this method, a proteinor peptide comprising the entirety or a segment of a marker protein issynthesized or isolated (e.g. by purification from a cell in which it isexpressed or by transcription and translation of a nucleic acid encodingthe protein or peptide in vivo or in vitro). A vertebrate, for example,a mammal such as a mouse, rat, rabbit, or sheep, is immunized using theprotein or peptide. The vertebrate may optionally (and preferably) beimmunized at least one additional time with the protein or peptide, sothat the vertebrate exhibits a robust immune response to the protein orpeptide. Splenocytes are isolated from the immunized vertebrate andfused with an immortalized cell line to form hybridomas, using any of avariety of methods. Hybridomas formed in this manner are then screenedusing standard methods to identify one or more hybridomas which producean antibody which specifically binds with the marker protein or afragment thereof. There is also provided herein hybridomas made by thismethod and antibodies made using such hybridomas.

Methods of Assessing Efficacy

There is also provided herein a method of assessing the efficacy of atest compound for inhibiting lung cancer-related disease cells. Asdescribed above, differences in the level of expression of the markerscorrelate with the abnormal state of lung cells. Although it isrecognized that changes in the levels of expression of certain of themarkers likely result from the abnormal state of lung cells, it islikewise recognized that changes in the levels of expression of other ofthe markers induce, maintain, and promote the abnormal state of thosecells. Thus, compounds which inhibit a lung cancer-related disease in apatient will cause the level of expression of one or more of the markersto change to a level nearer the normal level of expression for thatmarker (i.e. the level of expression for the marker in normal lungcells).

This method thus comprises comparing expression of a marker in a firstlung cell sample and maintained in the presence of the test compound andexpression of the marker in a second lung cell sample and maintained inthe absence of the test compound. A significantly reduced expression ofa marker in the presence of the test compound is an indication that thetest compound inhibits a lung cancer-related disease. The lung cellsamples may, for example, be aliquots of a single sample of normal lungcells obtained from a patient, pooled samples of normal lung cellsobtained from a patient, cells of a normal lung cell line, aliquots of asingle sample of lung cancer-related disease cells obtained from apatient, pooled samples of lung cancer-related disease cells obtainedfrom a patient, cells of a lung cancer-related disease cell line, or thelike.

In one embodiment, the samples are lung cancer-related disease cellsobtained from a patient and a plurality of compounds believed to beeffective for inhibiting various lung cancer-related diseases are testedin order to identify the compound which is likely to best inhibit thelung cancer-related disease in the patient.

This method may likewise be used to assess the efficacy of a therapy forinhibiting a lung cancer-related disease in a patient. In this method,the level of expression of one or more markers in a pair of samples (onesubjected to the therapy, the other not subjected to the therapy) isassessed. As with the method of assessing the efficacy of testcompounds, if the therapy induces a significantly lower level ofexpression of a marker then the therapy is efficacious for inhibiting alung cancer-related disease. As above, if samples from a selectedpatient are used in this method, then alternative therapies can beassessed in vitro in order to select a therapy most likely to beefficacious for inhibiting a lung cancer-related disease in the patient.

As described herein, the abnormal state of human lung cells iscorrelated with changes in the levels of expression of the markers.There is also provided a method for assessing the harmful potential of atest compound. This method comprises maintaining separate aliquots ofhuman lung cells in the presence and absence of the test compound.Expression of a marker in each of the aliquots is compared. Asignificantly higher level of expression of a marker in the aliquotmaintained in the presence of the test compound (relative to the aliquotmaintained in the absence of the test compound) is an indication thatthe test compound possesses a harmful potential. The relative harmfulpotential of various test compounds can be assessed by comparing thedegree of enhancement or inhibition of the level of expression of therelevant markers, by comparing the number of markers for which the levelof expression is enhanced or inhibited, or by comparing both.

Isolated Proteins and Antibodies

One aspect pertains to isolated marker proteins and biologically activeportions thereof, as well as polypeptide fragments suitable for use asimmunogens to raise antibodies directed against a marker protein or afragment thereof. In one embodiment, the native marker protein can beisolated from cells or tissue sources by an appropriate purificationscheme using standard protein purification techniques. In anotherembodiment, a protein or peptide comprising the whole or a segment ofthe marker protein is produced by recombinant DNA techniques.Alternative to recombinant expression, such protein or peptide can besynthesized chemically using standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theprotein is derived, or substantially free of chemical precursors orother chemicals when chemically synthesized. The language “substantiallyfree of cellular material” includes preparations of protein in which theprotein is separated from cellular components of the cells from which itis isolated or recombinantly produced. Thus, protein that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10%, or 5% (by dry weight) ofheterologous protein (also referred to herein as a “contaminatingprotein”).

When the protein or biologically active portion thereof is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, 10%, or 5% of thevolume of the protein preparation. When the protein is produced bychemical synthesis, it is preferably substantially free of chemicalprecursors or other chemicals, i.e., it is separated from chemicalprecursors or other chemicals which are involved in the synthesis of theprotein. Accordingly such preparations of the protein have less thanabout 30%, 20%, 10%, 5% (by dry weight) of chemical precursors orcompounds other than the polypeptide of interest.

Biologically active portions of a marker protein include polypeptidescomprising amino acid sequences sufficiently identical to or derivedfrom the amino acid sequence of the marker protein, which include feweramino acids than the full length protein, and exhibit at least oneactivity of the corresponding full-length protein. Typically,biologically active portions comprise a domain or motif with at leastone activity of the corresponding full-length protein. A biologicallyactive portion of a marker protein can be a polypeptide which is, forexample, 10, 25, 50, 100 or more amino acids in length. Moreover, otherbiologically active portions, in which other regions of the markerprotein are deleted, can be prepared by recombinant techniques andevaluated for one or more of the functional activities of the nativeform of the marker protein. In certain embodiments, useful proteins aresubstantially identical (e.g., at least about 40%, and in certainembodiments, 50%, 60%, 70%, 80%, 90%, 95%, or 99%) to one of thesesequences and retain the functional activity of the correspondingnaturally-occurring marker protein yet differ in amino acid sequence dueto natural allelic variation or mutagenesis.

In addition, libraries of segments of a marker protein can be used togenerate a variegated population of polypeptides for screening andsubsequent selection of variant marker proteins or segments thereof.

Predictive Medicine

There is also provided herein uses of the animal models and markers inthe field of predictive medicine in which diagnostic assays, prognosticassays, pharmacogenomics, and monitoring clinical trials are used forprognostic (predictive) purposes to thereby treat an individualprophylactically. Accordingly, there is also provided herein diagnosticassays for determining the level of expression of one or more markerproteins or nucleic acids, in order to determine whether an individualis at risk of developing a lung cancer-related disease. Such assays canbe used for prognostic or predictive purposes to therebyprophylactically treat an individual prior to the onset of the lungcancer-related disease.

In another aspect, the methods are useful for at least periodicscreening of the same individual to see if that individual has beenexposed to chemicals or toxins that change his/her expression patterns.

Yet another aspect pertains to monitoring the influence of agents (e.g.,drugs or other compounds administered either to inhibit a lungcancer-related disease or to treat or prevent any other disorder (e.g.,in order to understand any system effects that such treatment may have)on the expression or activity of a marker in clinical trials.

Pharmacogenomics

The markers are also useful as pharmacogenomic markers. As used herein,a “pharmacogenomic marker” is an objective biochemical marker whoseexpression level correlates with a specific clinical drug response orsusceptibility in a patient. The presence or quantity of thepharmacogenomic marker expression is related to the predicted responseof the patient and more particularly the patient's tumor to therapy witha specific drug or class of drugs. By assessing the presence or quantityof the expression of one or more pharmacogenomic markers in a patient, adrug therapy which is most appropriate for the patient, or which ispredicted to have a greater degree of success, may be selected.

Monitoring Clinical Trials

Monitoring the influence of agents (e.g., drug compounds) on the levelof expression of a marker can be applied not only in basic drugscreening, but also in clinical trials. For example, the effectivenessof an agent to affect marker expression can be monitored in clinicaltrials of subjects receiving treatment for a lung cancer-relateddisease.

In one non-limiting embodiment, the present invention provides a methodfor monitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate) comprising the steps of(i) obtaining a pre-administration sample from a subject prior toadministration of the agent; (ii) detecting the level of expression ofone or more selected markers in the pre-administration sample; (iii)obtaining one or more post-administration samples from the subject; (iv)detecting the level of expression of the marker(s) in thepost-administration samples; (v) comparing the level of expression ofthe marker(s) in the pre-administration sample with the level ofexpression of the marker(s) in the post-administration sample orsamples; and (vi) altering the administration of the agent to thesubject accordingly.

For example, increased expression of the marker gene(s) during thecourse of treatment may indicate ineffective dosage and the desirabilityof increasing the dosage. Conversely, decreased expression of the markergene(s) may indicate efficacious treatment and no need to change dosage.

Electronic Apparatus Readable Media, Systems, Arrays and Methods ofUsing Same

As used herein, “electronic apparatus readable media” refers to anysuitable medium for storing, holding or containing data or informationthat can be read and accessed directly by an electronic apparatus. Suchmedia can include, but are not limited to: magnetic storage media, suchas floppy discs, hard disc storage medium, and magnetic tape; opticalstorage media such as compact disc; electronic storage media such asRAM, ROM, EPROM, EEPROM and the like; and general hard disks and hybridsof these categories such as magnetic/optical storage media. The mediumis adapted or configured for having recorded thereon a marker asdescribed herein.

As used herein, the term “electronic apparatus” is intended to includeany suitable computing or processing apparatus or other deviceconfigured or adapted for storing data or information. Examples ofelectronic apparatus suitable for use with the present invention includestand-alone computing apparatus; networks, including a local areanetwork (LAN), a wide area network (WAN) Internet, Intranet, andExtranet; electronic appliances such as personal digital assistants(PDAs), cellular phone, pager and the like; and local and distributedprocessing systems.

As used herein, “recorded” refers to a process for storing or encodinginformation on the electronic apparatus readable medium. Those skilledin the art can readily adopt any method for recording information onmedia to generate materials comprising the markers described herein.

A variety of software programs and formats can be used to store themarker information of the present invention on the electronic apparatusreadable medium. Any number of data processor structuring formats (e.g.,text file or database) may be employed in order to obtain or create amedium having recorded thereon the markers. By providing the markers inreadable form, one can routinely access the marker sequence informationfor a variety of purposes. For example, one skilled in the art can usethe nucleotide or amino acid sequences in readable form to compare atarget sequence or target structural motif with the sequence informationstored within the data storage means. Search means are used to identifyfragments or regions of the sequences which match a particular targetsequence or target motif.

Thus, there is also provided herein a medium for holding instructionsfor performing a method for determining whether a subject has a lungcancer-related disease or a pre-disposition to a lung cancer-relateddisease, wherein the method comprises the steps of determining thepresence or absence of a marker and based on the presence or absence ofthe marker, determining whether the subject has a lung cancer-relateddisease or a pre-disposition to a lung cancer-related disease and/orrecommending a particular treatment for a lung cancer-related disease orpre-lung cancer-related disease condition.

There is also provided herein an electronic system and/or in a network,a method for determining whether a subject has a lung cancer-relateddisease or a pre-disposition to a lung cancer-related disease associatedwith a marker wherein the method comprises the steps of determining thepresence or absence of the marker, and based on the presence or absenceof the marker, determining whether the subject has a lung cancer-relateddisease or a pre-disposition to a lung cancer-related disease, and/orrecommending a particular treatment for the lung cancer-related diseaseor pre-lung cancer-related disease condition. The method may furthercomprise the step of receiving phenotypic information associated withthe subject and/or acquiring from a network phenotypic informationassociated with the subject.

Also provided herein is a network, a method for determining whether asubject has a lung cancer-related disease or a pre-disposition to a lungcancer-related disease associated with a marker, the method comprisingthe steps of receiving information associated with the marker, receivingphenotypic information associated with the subject, acquiringinformation from the network corresponding to the marker and/or a lungcancer-related disease, and based on one or more of the phenotypicinformation, the marker, and the acquired information, determiningwhether the subject has a lung cancer-related disease or apre-disposition to a lung cancer-related disease. The method may furthercomprise the step of recommending a particular treatment for the lungcancer-related disease or pre-lung cancer-related disease condition.

There is also provided herein a business method for determining whethera subject has a lung cancer-related disease or a pre-disposition to alung cancer-related disease, the method comprising the steps ofreceiving information associated with the marker, receiving phenotypicinformation associated with the subject, acquiring information from thenetwork corresponding to the marker and/or a lung cancer-relateddisease, and based on one or more of the phenotypic information, themarker, and the acquired information, determining whether the subjecthas a lung cancer-related disease or a pre-disposition to a lungcancer-related disease. The method may further comprise the step ofrecommending a particular treatment for the lung cancer-related diseaseor pre-lung cancer-related disease condition.

There is also provided herein an array that can be used to assayexpression of one or more genes in the array. In one embodiment, thearray can be used to assay gene expression in a tissue to ascertaintissue specificity of genes in the array. In this manner, up to about7000 or more genes can be simultaneously assayed for expression. Thisallows a profile to be developed showing a battery of genes specificallyexpressed in one or more tissues.

In addition to such qualitative determination, there is provided hereinthe quantitation of gene expression. Thus, not only tissue specificity,but also the level of expression of a battery of genes in the tissue isascertainable. Thus, genes can be grouped on the basis of their tissueexpression per se and level of expression in that tissue. This isuseful, for example, in ascertaining the relationship of gene expressionbetween or among tissues. Thus, one tissue can be perturbed and theeffect on gene expression in a second tissue can be determined. In thiscontext, the effect of one cell type on another cell type in response toa biological stimulus can be determined.

Such a determination is useful, for example, to know the effect ofcell-cell interaction at the level of gene expression. If an agent isadministered therapeutically to treat one cell type but has anundesirable effect on another cell type, the method provides an assay todetermine the molecular basis of the undesirable effect and thusprovides the opportunity to co-administer a counteracting agent orotherwise treat the undesired effect. Similarly, even within a singlecell type, undesirable biological effects can be determined at themolecular level. Thus, the effects of an agent on expression of otherthan the target gene can be ascertained and counteracted.

In another embodiment, the array can be used to monitor the time courseof expression of one or more genes in the array. This can occur invarious biological contexts, as disclosed herein, for exampledevelopment of a lung cancer-related disease, progression of a lungcancer-related disease, and processes, such as cellular transformationassociated with a lung cancer-related disease.

The array is also useful for ascertaining the effect of the expressionof a gene or the expression of other genes in the same cell or indifferent cells. This provides, for example, for a selection ofalternate molecular targets for therapeutic intervention if the ultimateor downstream target cannot be regulated.

The array is also useful for ascertaining differential expressionpatterns of one or more genes in normal and abnormal cells. Thisprovides a battery of genes that could serve as a molecular target fordiagnosis or therapeutic intervention.

Surrogate Markers

The markers may serve as surrogate markers for one or more disorders ordisease states or for conditions leading up to a lung cancer-relateddisease state. As used herein, a “surrogate marker” is an objectivebiochemical marker which correlates with the absence or presence of adisease or disorder, or with the progression of a disease or disorder.The presence or quantity of such markers is independent of the disease.Therefore, these markers may serve to indicate whether a particularcourse of treatment is effective in lessening a disease state ordisorder. Surrogate markers are of particular use when the presence orextent of a disease state or disorder is difficult to assess throughstandard methodologies, or when an assessment of disease progression isdesired before a potentially dangerous clinical endpoint is reached.

The markers are also useful as pharmacodynamic markers. As used herein,a “pharmacodynamic marker” is an objective biochemical marker whichcorrelates specifically with drug effects. The presence or quantity of apharmacodynamic marker is not related to the disease state or disorderfor which the drug is being administered; therefore, the presence orquantity of the marker is indicative of the presence or activity of thedrug in a subject. For example, a pharmacodynamic marker may beindicative of the concentration of the drug in a biological tissue, inthat the marker is either expressed or transcribed or not expressed ortranscribed in that tissue in relationship to the level of the drug. Inthis fashion, the distribution or uptake of the drug may be monitored bythe pharmacodynamic marker. Similarly, the presence or quantity of thepharmacodynamic marker may be related to the presence or quantity of themetabolic product of a drug, such that the presence or quantity of themarker is indicative of the relative breakdown rate of the drug in vivo.

Pharmacodynamic markers are of particular use in increasing thesensitivity of detection of drug effects, particularly when the drug isadministered in low doses. Since even a small amount of a drug may besufficient to activate multiple rounds of marker transcription orexpression, the amplified marker may be in a quantity which is morereadily detectable than the drug itself. Also, the marker may be moreeasily detected due to the nature of the marker itself; for example,using the methods described herein, antibodies may be employed in animmune-based detection system for a protein marker, or marker-specificradiolabeled probes may be used to detect a mRNA marker. Furthermore,the use of a pharmacodynamic marker may offer mechanism-based predictionof risk due to drug treatment beyond the range of possible directobservations.

Protocols for Testing

The method of testing for lung cancer-related diseases comprises, forexample measuring the expression level of each marker gene in abiological sample from a subject over time and comparing the level withthat of the marker gene in a control biological sample.

When the marker gene is one of the genes described herein and theexpression level is differentially expressed (for examples, higher orlower than that in the control), the subject is judged to be affectedwith a lung cancer-related disease. When the expression level of themarker gene falls within the permissible range, the subject is unlikelyto be affected with a lung cancer-related disease.

The standard value for the control may be pre-determined by measuringthe expression level of the marker gene in the control, in order tocompare the expression levels. For example, the standard value can bedetermined based on the expression level of the above-mentioned markergene in the control. For example, in certain embodiments, thepermissible range is taken as ±2 S.D. based on the standard value. Oncethe standard value is determined, the testing method may be performed bymeasuring only the expression level in a biological sample from asubject and comparing the value with the determined standard value forthe control.

Expression levels of marker genes include transcription of the markergenes to mRNA, and translation into proteins. Therefore, one method oftesting for a lung cancer-related disease is performed based on acomparison of the intensity of expression of mRNA corresponding to themarker genes, or the expression level of proteins encoded by the markergenes.

The measurement of the expression levels of marker genes in the testingfor a lung cancer-related disease can be carried out according tovarious gene analysis methods. Specifically, one can use, for example, ahybridization technique using nucleic acids that hybridize to thesegenes as probes, or a gene amplification technique using DNA thathybridize to the marker genes as primers.

The probes or primers used for the testing can be designed based on thenucleotide sequences of the marker genes. The identification numbers forthe nucleotide sequences of the respective marker genes are describerherein.

Further, it is to be understood that genes of higher animals generallyaccompany polymorphism in a high frequency. There are also manymolecules that produce isoforms comprising mutually different amino acidsequences during the splicing process. Any gene associated with a lungcancer-related disease that has an activity similar to that of a markergene is included in the marker genes, even if it has nucleotide sequencedifferences due to polymorphism or being an isoform.

It is also to be understood that the marker genes can include homologsof other species in addition to humans. Thus, unless otherwisespecified, the expression “marker gene” refers to a homolog of themarker gene unique to the species or a foreign marker gene which hasbeen introduced into an individual.

Also, it is to be understood that a “homolog of a marker gene” refers toa gene derived from a species other than a human, which can hybridize tothe human marker gene as a probe under stringent conditions. Suchstringent conditions are known to one skilled in the art who can selectan appropriate condition to produce an equal stringency experimentallyor empirically.

A polynucleotide comprising the nucleotide sequence of a marker gene ora nucleotide sequence that is complementary to the complementary strandof the nucleotide sequence of a marker gene and has at least 15nucleotides, can be used as a primer or probe. Thus, a “complementarystrand” means one strand of a double stranded DNA with respect to theother strand and which is composed of A:T (U for RNA) and G:C basepairs.

In addition, “complementary” means not only those that are completelycomplementary to a region of at least 15 continuous nucleotides, butalso those that have a nucleotide sequence homology of at least 40% incertain instances, 50% in certain instances, 60% in certain instances,70% in certain instances, at least 80%, 90%, and 95% or higher. Thedegree of homology between nucleotide sequences can be determined by analgorithm, BLAST, etc.

Such polynucleotides are useful as a probe to detect a marker gene, oras a primer to amplify a marker gene. When used as a primer, thepolynucleotide comprises usually 15 bp to 100 bp, and in certainembodiments 15 bp to 35 bp of nucleotides. When used as a probe, a DNAcomprises the whole nucleotide sequence of the marker gene (or thecomplementary strand thereof), or a partial sequence thereof that has atleast 15 bp nucleotides. When used as a primer, the 3′ region must becomplementary to the marker gene, while the 5′ region can be linked to arestriction enzyme-recognition sequence or a tag.

“Polynucleotides” may be either DNA or RNA. These polynucleotides may beeither synthetic or naturally-occurring. Also, DNA used as a probe forhybridization is usually labeled. Those skilled in the art readilyunderstand such labeling methods. Herein, the term “oligonucleotide”means a polynucleotide with a relatively low degree of polymerization.Oligonucleotides are included in polynucleotides.

Tests for a lung cancer-related disease using hybridization techniquescan be performed using, for example, Northern hybridization, dot blothybridization, or the DNA microarray technique. Furthermore, geneamplification techniques, such as the RT-PCR method may be used. Byusing the PCR amplification monitoring method during the geneamplification step in RT-PCR, one can achieve a more quantitativeanalysis of the expression of a marker gene.

In the PCR gene amplification monitoring method, the detection target(DNA or reverse transcript of RNA) is hybridized to probes that arelabeled with a fluorescent dye and a quencher which absorbs thefluorescence. When the PCR proceeds and Taq polymerase degrades theprobe with its 5′-3′ exonuclease activity, the fluorescent dye and thequencher draw away from each other and the fluorescence is detected. Thefluorescence is detected in real time. By simultaneously measuring astandard sample in which the copy number of a target is known, it ispossible to determine the copy number of the target in the subjectsample with the cycle number where PCR amplification is linear. Also,one skilled in the art recognizes that the PCR amplification monitoringmethod can be carried out using any suitable method.

The method of testing for a lung cancer-related disease can be alsocarried out by detecting a protein encoded by a marker gene.Hereinafter, a protein encoded by a marker gene is described as a“marker protein.” For such test methods, for example, the Westernblotting method, the immunoprecipitation method, and the ELISA methodmay be employed using an antibody that binds to each marker protein.

Antibodies used in the detection that bind to the marker protein may beproduced by any suitable technique. Also, in order to detect a markerprotein, such an antibody may be appropriately labeled. Alternatively,instead of labeling the antibody, a substance that specifically binds tothe antibody, for example, protein A or protein G, may be labeled todetect the marker protein indirectly. More specifically, such adetection method can include the ELISA method.

A protein or a partial peptide thereof used as an antigen may beobtained, for example, by inserting a marker gene or a portion thereofinto an expression vector, introducing the construct into an appropriatehost cell to produce a transformant, culturing the transformant toexpress the recombinant protein, and purifying the expressed recombinantprotein from the culture or the culture supernatant. Alternatively, theamino acid sequence encoded by a gene or an oligopeptide comprising aportion of the amino acid sequence encoded by a full-length cDNA arechemically synthesized to be used as an immunogen.

Furthermore, a test for a lung cancer-related disease can be performedusing as an index not only the expression level of a marker gene butalso the activity of a marker protein in a biological sample. Activityof a marker protein means the biological activity intrinsic to theprotein. Various methods can be used for measuring the activity of eachprotein.

Even if a patient is not diagnosed as being affected with a lungcancer-related disease in a routine test in spite of symptoms suggestingthese diseases, whether or not such a patient is suffering from a lungcancer-related disease can be easily determined by performing a testaccording to the methods described herein.

More specifically, in certain embodiments, when the marker gene is oneof the genes described herein, an increase or decrease in the expressionlevel of the marker gene in a patient whose symptoms suggest at least asusceptibility to a lung cancer-related disease indicates that thesymptoms are primarily caused by a lung cancer-related disease.

In addition, the tests are useful to determine whether a lungcancer-related disease is improving in a patient. In other words, themethods described herein can be used to judge the therapeutic effect ofa treatment for a lung cancer-related disease. Furthermore, when themarker gene is one of the genes described herein, an increase ordecrease in the expression level of the marker gene in a patient, whohas been diagnosed as being affected by a lung cancer-related disease,implies that the disease has progressed more.

The severity and/or susceptibility to a lung cancer-related disease mayalso be determined based on the difference in expression levels. Forexample, when the marker gene is one of the genes described herein, thedegree of increase in the expression level of the marker gene iscorrelated with the presence and/or severity of a lung cancer-relateddisease.

In addition, the expression itself of a marker gene can be controlled byintroducing a mutation(s) into the transcriptional regulatory region ofthe gene. Those skilled in the art understand such amino acidsubstitutions. Also, the number of amino acids that are mutated is notparticularly restricted, as long as the activity is maintained Normally,it is within 50 amino acids, in certain non-limiting embodiments, within30 amino acids, within 10 amino acids, or within 3 amino acids. The siteof mutation may be any site, as long as the activity is maintained.

In yet another aspect, there is provided herein screening methods forcandidate compounds for therapeutic agents to treat a lungcancer-related disease. One or more marker genes are selected from thegroup of genes described herein. A therapeutic agent for a lungcancer-related disease can be obtained by selecting a compound capableof increasing or decreasing the expression level of the marker gene(s).

It is to be understood that the expression “a compound that increasesthe expression level of a gene” refers to a compound that promotes anyone of the steps of gene transcription, gene translation, or expressionof a protein activity. On the other hand, the expression “a compoundthat decreases the expression level of a gene”, as used herein, refersto a compound that inhibits any one of these steps.

In particular aspects, the method of screening for a therapeutic agentfor a lung cancer-related disease can be carried out either in vivo orin vitro. This screening method can be performed, for example, by (1)administering a candidate compound to an animal subject; (2) measuringthe expression level of a marker gene(s) in a biological sample from theanimal subject; or (3) selecting a compound that increases or decreasesthe expression level of a marker gene(s) as compared to that in acontrol with which the candidate compound has not been contacted.

In still another aspect, there is provided herein a method to assess theefficacy of a candidate compound for a pharmaceutical agent on theexpression level of a marker gene(s) by contacting an animal subjectwith the candidate compound and monitoring the effect of the compound onthe expression level of the marker gene(s) in a biological samplederived from the animal subject. The variation in the expression levelof the marker gene(s) in a biological sample derived from the animalsubject can be monitored using the same technique as used in the testingmethod described above. Furthermore, based on the evaluation, acandidate compound for a pharmaceutical agent can be selected byscreening.

Kits

In another aspect, there is provided various diagnostic and test kits.In one embodiment, a kit is useful for assessing whether a patient isafflicted with a lung cancer-related disease. The kit comprises areagent for assessing expression of a marker. In another embodiment, akit is useful for assessing the suitability of a chemical or biologicagent for inhibiting a lung cancer-related disease in a patient. Such akit comprises a reagent for assessing expression of a marker, and mayalso comprise one or more of such agents.

In a further embodiment, the kits are useful for assessing the presenceof lung cancer-related disease cells or treating lung cancer-relateddiseases. Such kits comprise an antibody, an antibody derivative or anantibody fragment, which binds specifically with a marker protein or afragment of the protein. Such kits may also comprise a plurality ofantibodies, antibody derivatives or antibody fragments wherein theplurality of such antibody agents binds specifically with a markerprotein or a fragment of the protein.

In an additional embodiment, the kits are useful for assessing thepresence of lung cancer-related disease cells, wherein the kit comprisesa nucleic acid probe that binds specifically with a marker nucleic acidor a fragment of the nucleic acid. The kit may also comprise a pluralityof probes, wherein each of the probes binds specifically with a markernucleic acid, or a fragment of the nucleic acid.

The compositions, kits and methods described herein can have thefollowing uses, among others: 1) assessing whether a patient isafflicted with a lung cancer-related disease; 2) assessing the stage ofa lung cancer-related disease in a human patient; 3) assessing the gradeof a lung cancer-related disease in a patient; 4) assessing the natureof a lung cancer-related disease in a patient; 5) assessing thepotential to develop a lung cancer-related disease in a patient; 6)assessing the histological type of cells associated with a lungcancer-related disease in a patient; 7) making antibodies, antibodyfragments or antibody derivatives that are useful for treating a lungcancer-related disease and/or assessing whether a patient is afflictedwith a lung cancer-related disease; 8) assessing the presence of lungcancer-related disease cells; 9) assessing the efficacy of one or moretest compounds for inhibiting a lung cancer-related disease in apatient; 10) assessing the efficacy of a therapy for inhibiting a lungcancer-related disease in a patient; 11) monitoring the progression of alung cancer-related disease in a patient; 12) selecting a composition ortherapy for inhibiting a lung cancer-related disease in a patient; 13)treating a patient afflicted with a lung cancer-related disease; 14)inhibiting a lung cancer-related disease in a patient; 15) assessing theharmful potential of a test compound; and 16) preventing the onset of alung cancer-related disease in a patient at risk for developing a lungcancer-related disease.

The kits are useful for assessing the presence of lung cancer-relateddisease cells (e.g. in a sample such as a patient sample). The kitcomprises a plurality of reagents, each of which is capable of bindingspecifically with a marker nucleic acid or protein. Suitable reagentsfor binding with a marker protein include antibodies, antibodyderivatives, antibody fragments, and the like. Suitable reagents forbinding with a marker nucleic acid (e.g. a genomic DNA, an mRNA, aspliced mRNA, a cDNA, or the like) include complementary nucleic acids.For example, the nucleic acid reagents may include oligonucleotides(labeled or non-labeled) fixed to a substrate, labeled oligonucleotidesnot bound with a substrate, pairs of PCR primers, molecular beaconprobes, and the like.

The kits may optionally comprise additional components useful forperforming the methods described herein. By way of example, the kit maycomprise fluids (e.g. SSC buffer) suitable for annealing complementarynucleic acids or for binding an antibody with a protein with which itspecifically binds, one or more sample compartments, an instructionalmaterial which describes performance of the method, a sample of normallung cells, a sample of lung cancer-related disease cells, and the like.

Animal Model

In a broad aspect, there is provided a method for producing a non-humananimal model for assessment of at least one lung cancer-related disease.The method includes exposing the animal to repeated doses of at leastone chemical believed to cause lung cancer. In certain aspects, themethod further includes collecting one or more selected samples from theanimal; and comparing the collected sample to one or more indicia ofpotential lung cancer initiation or development.

In a broad aspect, there is provides a method of producing the animalmodel that includes: maintaining the animal in a specific chemical-freeenvironment and sensitizing the animal with at least one chemicalbelieved to cause lung cancer. In certain embodiments, at least a partof the animal's lung is sensitized by multiple sequential exposures. Inanother broad aspect, there is provided a method of screening for anagent for effectiveness against at least one lung cancer-relateddisease. The method generally includes: administering at least one agentto a test animal, determining whether the agent reduces or aggravatesone or more symptoms of the lung cancer-related disease; correlating areduction in one or more symptoms with effectiveness of the agentagainst the lung cancer-related disease; or correlating a lack ofreduction in one or more symptoms with ineffectiveness of the agent. Theanimal model is useful for assessing one or more metabolic pathways thatcontribute to at least one of initiation, progression, severity,pathology, aggressiveness, grade, activity, disability, mortality,morbidity, disease sub-classification or other underlying pathogenic orpathological feature of at least one lung cancer-related disease. Theanalysis can be by one or more of: hierarchical clustering, signaturenetwork construction, mass spectroscopy proteomic analysis, surfaceplasmon resonance, linear statistical modeling, partial least squaresdiscriminant analysis, and multiple linear regression analysis.

In a particular aspect, the animal model is assessed for at least onelung cancer-related disease, by examining an expression level of one ormore markers, or a functional equivalent thereto.

The animal models created by the methods described herein will enablescreening of therapeutic agents useful for treating or preventing a lungcancer-related disease. Accordingly, the methods are useful foridentifying therapeutic agents for treating or preventing a lungcancer-related disease. The methods comprise administering a candidateagent to an animal model made by the methods described herein, assessingat least one lung cancer-related disease response in the animal model ascompared to a control animal model to which the candidate agent has notbeen administered. If at least one lung cancer-related disease responseis reduced in symptoms or delayed in onset, the candidate agent is anagent for treating or preventing the lung cancer-related disease.

In another aspect, there is provided herein animal models for a lungcancer-related disease where the expression level of one or more markergenes or a gene functionally equivalent to the marker gene has beenelevated in the animal model. A “functionally equivalent gene” as usedherein generally is a gene that encodes a protein having an activitysimilar to a known activity of a protein encoded by the marker gene. Arepresentative example of a functionally equivalent gene includes acounterpart of a marker gene of a subject animal, which is intrinsic tothe animal.

The animal model for a lung cancer-related disease is useful fordetecting physiological changes due to a lung cancer-related disease. Incertain embodiments, the animal model is useful to reveal additionalfunctions of marker genes and to evaluate drugs whose targets are themarker genes.

In one embodiment, an animal model for a lung cancer-related disease canbe created by controlling the expression level of a counterpart gene oradministering a counterpart gene. The method can include creating ananimal model for a lung cancer-related disease by controlling theexpression level of a gene selected from the group of genes describedherein. In another embodiment, the method can include creating an animalmodel for a lung cancer-related disease by administering the proteinencoded by a gene described herein, or administering an antibody againstthe protein. It is to be also understood, that in certain otherembodiments, the marker can be over-expressed such that the marker canthen be measured using appropriate methods.

In another embodiment, an animal model for a lung cancer-related diseasecan be created by introducing a gene selected from such groups of genes,or by administering a protein encoded by such a gene.

In another embodiment, a lung cancer-related disease can be induced bysuppressing the expression of a gene selected from such groups of genesor the activity of a protein encoded by such a gene. An antisensenucleic acid, a ribozyme, or an RNAi can be used to suppress theexpression. The activity of a protein can be controlled effectively byadministering a substance that inhibits the activity, such as anantibody.

The animal model is useful to elucidate the mechanism underlying a lungcancer-related disease and also to test the safety of compounds obtainedby screening. For example, when an animal model develops the symptoms oflung cancer-related disease, or when a measured value involved in acertain a lung cancer-related disease alters in the animal, a screeningsystem can be constructed to explore compounds having activity toalleviate the disease.

As used herein, the expression “an increase in the expression level”refers to any one of the following: where a marker gene introduced as aforeign gene is expressed artificially; where the transcription of amarker gene intrinsic to the subject animal and the translation thereofinto the protein are enhanced; or where the hydrolysis of the protein,which is the translation product, is suppressed. As used herein, theexpression “a decrease in the expression level” refers to either thestate in which the transcription of a marker gene of the subject animaland the translation thereof into the protein are inhibited, or the statein which the hydrolysis of the protein, which is the translationproduct, is enhanced. The expression level of a gene can be determined,for example, by a difference in signal intensity on a DNA chip.Furthermore, the activity of the translation product—the protein—can bedetermined by comparing with that in the normal state.

It is also within the contemplated scope that the animal model caninclude transgenic animals, including, for example animals where amarker gene has been introduced and expressed artificially; marker geneknockout animals; and knock-in animals in which another gene has beensubstituted for a marker gene. A transgenic animal, into which anantisense nucleic acid of a marker gene, a ribozyme, a polynucleotidehaving an RNAi effect, or a DNA functioning as a decoy nucleic acid orsuch has been introduced, can be used as the transgenic animal. Suchtransgenic animals also include, for example, animals in which theactivity of a marker protein has been enhanced or suppressed byintroducing a mutation(s) into the coding region of the gene, or theamino acid sequence has been modified to become resistant or susceptibleto hydrolysis. Mutations in an amino acid sequence includesubstitutions, deletions, insertions, and additions.

All patents, patent applications and references cited herein areincorporated in their entirety by reference. While the invention hasbeen described and exemplified in sufficient detail for those skilled inthis art to make and use it, various alternatives, modifications andimprovements should be apparent without departing from the spirit andscope of the invention. One skilled in the art readily appreciates thatthe present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those inherenttherein.

The methods and reagents described herein are representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Modifications therein andother uses will occur to those skilled in the art. These modificationsare encompassed within the spirit of the invention and are defined bythe scope of the claims. It will also be readily apparent to a personskilled in the art that varying substitutions and modifications may bemade to the invention disclosed herein without departing from the scopeand spirit of the invention.

It should be understood that although the present invention has beenspecifically disclosed by preferred embodiments and optional features,modifications and variations of the concepts herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention asdefined by the appended claims.

While the invention has been described with reference to various andpreferred embodiments, it should be understood by those skilled in theart that various changes may be made and equivalents may be substitutedfor elements thereof without departing from the essential scope of theinvention. In addition, many modifications may be made to adapt aparticular situation or material to the teachings of the inventionwithout departing from the essential scope thereof.

Therefore, it is intended that the invention not be limited to theparticular embodiment disclosed herein contemplated for carrying outthis invention, but that the invention will include all embodimentsfalling within the scope of the claims.

The publication and other material used herein to illuminate theinvention or provide additional details respecting the practice of theinvention, are incorporated be reference herein, and for convenience areprovided in the following bibliography.

Citation of the any of the documents recited herein is not intended asan admission that any of the foregoing is pertinent prior art. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicant anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

What is claimed is:
 1. A pharmaceutical composition for treating a lungcancer-related disease, comprising: at least one miR gene productselected from the group consisting of miR-29a, miR-29b, miR-29c andcombinations thereof; and, a pharmaceutically-acceptable carrier.
 2. Thepharmaceutical composition of claim 1, wherein the at least one miR geneproduct corresponds to a miR gene product that is down-regulated incancer cells relative to suitable control cells.
 3. The pharmaceuticalcomposition of claim 1, wherein the lung cancer-related disease isnon-small cell lung cancer (NSCLC).
 4. A composition used to treatcancer in a patient, the composition comprising an inhibitor of one ormore of DNMT1, DNMT3A and/or DNMT3B, wherein the inhibitor comprises oneor more miR-29 gene products, and wherein the cancer is characterized asexhibiting an undesirable DNA methylation pattern.
 5. The composition ofclaim 4, wherein the miR-29 gene product comprises to a miRNA that hasless than 100% identity to a corresponding wild-type miR gene productand possesses one or more biological activities of the correspondingwild-type miR gene product.
 6. The composition of claim 4, wherein themiR-29 gene product includes species variants and variants that are theconsequence of one or more mutations in a miR gene.
 7. The compositionof claim 4, wherein the miR-29 gene product is at least about 70%, 75%,80%, 85%, 90%, 95%, 98%, or 99% identical to a corresponding wild-typemiR gene product.
 8. The composition of claim 4, wherein the miR-29 geneproduct is an oligonucleotide that binds to miR-29.
 9. The compositionof claim 4, wherein the miR-29 gene product physically interacts withmiR-29.
 10. A pharmaceutical composition comprising a nucleic acid thatencodes a miRNA having an inhibitory effect on expression of one or moreof DNMT1, DNMT3a and/or DNMT3B in an amount effective to treat a cancer.11. The pharmaceutical composition of claim 10, wherein the miRNA is amature miRNA, a precursor of the miRNA, or variants and analogs thereof.12. The pharmaceutical composition of claim 10, wherein the miRNA is asubstance comprising a seed sequence of SEQ ID NO: 1 (hsa-miR-29a), SEQID NO: 2 (hsa-miR-29b) or SEQ ID NO: 3 (hsa-miR-29c), precursorsthereof, or variants and analogs thereof.
 13. The pharmaceuticalcomposition of claim 10, wherein the miRNA is comprised within anexpression vector for introducing and expressing in cancer cells.
 14. Amethod of diagnosing whether a subject has a decreases survivalprognosis for a lung cancer-related disease, comprising: measuring thelevel of DNMT3A in a test sample from the subject, wherein an increasein the level of DNMT3A in the test sample, relative to the level of acorresponding DNMT3A in a control sample, is indicative of the subjecteither having an increased risk for a poor survival prognosis.
 15. Amethod for restoring a desired pattern of DNA methylation in a subjectin need thereof, wherein the method comprises: administering to thesubject an effective amount of one or more miR-29s sufficient to targetat least one methyltransferase comprising DNMT1, DNMT3A, and/or DNMT3B;and restoring the desired pattern of DNA methylation in the subject,wherein the one or more miR-29s comprise at least one isolated orsynthetic miR-29 selected from the group consisting of miR-29a, miR-29b,and miR-29c, and wherein the subject has an undesirable pattern of DNAmethylation.
 16. A method for inducing re-expression of at least onemethylation-silenced tumor suppressor gene (TSG) in a subject in needthereof, wherein the method comprises: administering to the subject aneffective amount of one or more miR-29s sufficient to target at leastone methyltransferase comprising DNMT1, DNMT3A, and/or DNMT3B; andinducing re-expression of the at least one methylation-silenced TSG inthe subject, wherein the one or more miR-29s comprise at least oneisolated or synthetic miR-29 selected from the group consisting ofmiR-29a, miR-29b, and miR-29c, and wherein the subject has anundesirable decreased expression of at least one TSG.
 17. A method forinducing re-expression of at least one methylation-silenced tumorsuppressor gene (TSG) in a cell in need thereof, wherein the methodcomprises: transfecting the cell with an effective amount of one or moremiR-29s sufficient to target at least one methyltransferase comprisingDNMT1, DNMT3A, and/or DNMT3B; and inducing re-expression of the at leastone methylation-silenced TSG in the cell, wherein the one or moremiR-29s comprise at least one isolated or synthetic miR-29 selected fromthe group consisting of miR-29a, miR-29b, and miR-29c, and wherein thecell is a cancer cell having an increased expression of one or more ofDNMT1, DNMT3a and/or DNMT3B.
 18. A method for inhibiting tumorigenicityin a subject in need thereof, wherein the method comprises:administering to the subject an effective amount of one or more miR-29ssufficient to target at least one methyltransferase comprising DNMT1,DNMT3A, and/or DNMT3B; and inhibiting tumorigenicity in the subject,wherein the one or more miR-29s comprise at least one isolated orsynthetic miR-29 selected from the group consisting of miR-29a, miR-29b,and miR-29c, and wherein the subject has an increased expression of oneor more of DNMT1, DNMT3a and/or DNMT3B.
 19. A method for inhibitingtumorigenicity in a cell in need thereof, wherein the method comprises:transfecting the cell with an effective amount of one or more miR-29ssufficient to target at least one methyltransferase comprising DNMT1,DNMT3A, and/or DNMT3B; and inhibiting tumorigenicity in the cell,wherein the one or more miR-29s comprise at least one isolated orsynthetic miR-29 selected from the group consisting of miR-29a, miR-29b,and miR-29c, and wherein the cell is a cancer cell having an increasedexpression of one or more of DNMT1, DNMT3a and/or DNMT3B.
 20. A methodfor reducing global DNA methylation in a subject in need thereof,wherein the method comprises: administering to the subject an effectiveamount of one or more miR-29s sufficient to target at least onemethyltransferase comprising DNMT1, DNMT3A, and/or DNMT3B; and reducingglobal DNA methylation in the subject, wherein the one or more miR-29scomprise at least one isolated or synthetic miR-29 selected from thegroup consisting of miR-29a, miR-29b, and miR-29c, and wherein thesubject has a cancer having an increased expression of one or more ofDNMT1, DNMT3a and/or DNMT3B.
 21. The method of claim 20, wherein thereducing global DNA methylation includes DNA epigenetic regulation ofthe cancer by up-regulating expression of the one or more miR-29s in thesubject.
 22. The method of claim 20, wherein the administering includescombining the one or more miR-29s with an effective amount of one ormore nucleoside analogs sufficient to block at least one de novo andmaintenance methyltransferase (DNMT) pathway.
 23. The method of claim22, wherein the one or more nucleoside analogs comprise decitabine. 24.A method for reducing global DNA methylation in a cell in need thereof,wherein the method comprises: transfecting the cell with an effectiveamount of one or more miR-29s sufficient to target at least onemethyltransferase comprising DNMT1, DNMT3A, and/or DNMT3B; and reducingglobal DNA methylation in the cell, wherein the one or more miR-29scomprise at least one isolated or synthetic miR-29 selected from thegroup consisting of miR-29a, miR-29b, and miR-29c, and wherein the cellis a cancer cell having an increased expression of one or more of DNMT1,DNMT3a and/or DNMT3B.
 25. The method of claim 24, wherein the reducingglobal DNA methylation includes DNA epigenetic modification of the cellby up-regulating expression of the one or more miR-29s in the cancercell.
 26. The method of claim 25, wherein the transfecting includescombining the one or more miR-29s with an effective amount of one ormore nucleoside analogs sufficient to block at least one de novo andmaintenance methyltransferase (DNMT) pathway.
 27. The method of claim26, wherein the one or more nucleoside analogs comprise decitabine. 28.A method for increasing expression of at least one tumor suppressiongene (TSG) in a subject in need thereof, wherein the method comprises:administering to the subject an effective amount of one or more miR-29ssufficient to target at least one methyltransferase comprising DNMT1,DNMT3A, and/or DNMT3B; and increasing expression of the at least one TSGin the subject, wherein the one or more miR-29s comprise at least oneisolated or synthetic miR-29 selected from the group consisting ofmiR-29a, miR-29b, and miR-29c, and wherein the subject has a cancerhaving an increased expression of one or more of DNMT1, DNMT3a and/orDNMT3B.
 29. The method of claim 28, wherein the at least one TSGcomprises FHIT and/or WWOX.
 30. A method for increasing expression of atleast one tumor suppression gene (TSG) in a cell in need thereof,wherein the method comprises: transfecting the cell with an effectiveamount of one or more miR-29s sufficient to target at least onemethyltransferase comprising DNMT1, DNMT3A, and/or DNMT3B; andincreasing expression of the at least one TSG in the cell, wherein theone or more miR-29s comprise at least one isolated or synthetic miR-29selected from the group consisting of miR-29a, miR-29b, and miR-29c, andwherein the cell is a cancer cell having an increased expression of oneor more of DNMT1, DNMT3a and/or DNMT3B.
 31. The method of claim 30,wherein the at least one TSG comprises FHIT and/or WWOX.
 32. A methodfor increasing expression of at least one FHIT and/or WWOX enzyme in asubject in need thereof, wherein the method comprises: administering tothe subject an effective amount of one or more miR-29s sufficient todecrease expression of at least one methyltransferase comprising DNMT1,DNMT3A, and/or DNMT3B; and increasing expression of the at least oneFHIT and/or WWOX enzyme in the subject, wherein the one or more miR-29scomprise at least one isolated or synthetic miR-29 selected from thegroup consisting of miR-29a, miR-29b, and miR-29c, and wherein thesubject has a cancer having an increased expression of one or more ofDNMT1, DNMT3a and/or DNMT3B.
 33. A method for increasing expression ofat least one FHIT and/or WWOX enzyme in a cell in need thereof, whereinthe method comprises: transfecting the cell with an effective amount ofone or more miR-29s sufficient to decrease expression of at least onemethyltransferase comprising DNMT1, DNMT3A, and/or DNMT3B; andincreasing expression of at least one FHIT and/or WWOX enzyme in thecell, wherein the one or more miR-29s comprise at least one isolated orsynthetic miR-29 selected from the group consisting of miR-29a, miR-29b,and miR-29c, and wherein the cell is a cancer cell an increasedexpression of one or more of DNMT1, DNMT3a and/or DNMT3B.
 34. A methodfor reducing global DNA methylation in a subject in need thereof,wherein the method comprises: administering to the subject an effectiveamount of one or more miR-29s sufficient to decrease expression of atleast one methyltransferase comprising DNMT1, DNMT3A, and/or DNMT3B; andreducing global DNA methylation in the subject, wherein the reducingglobal DNA methylation includes inducing expression of the one or moremiR-29s in the subject, wherein the one or more miR-29s comprise atleast one isolated or synthetic miR-29 selected from the groupconsisting of miR-29a, miR-29b, and miR-29c, and wherein the subject hasa cancer having an increased expression of one or more of DNMT1, DNMT3aand/or DNMT3B.
 35. A method for reducing global DNA methylation in acell in need thereof, wherein the method comprises: transfecting thecell with an effective amount of one or more miR-29s sufficient todecrease expression of at least one methyltransferase comprising DNMT1,DNMT3A, and/or DNMT3B; and reducing global DNA methylation in the cell,wherein the reducing global DNA methylation includes inducing expressionof the one or more miR-29s in the cell, wherein the one or more miR-29scomprise at least one isolated or synthetic miR-29 selected from thegroup consisting of miR-29a, miR-29b, and miR-29c, and wherein the cellis a cancer cell having an increased expression of one or more of DNMT1,DNMT3a and/or DNMT3B.
 36. A method for restoring expression of at leastone tumor suppressor gene (TSG) in a subject in need thereof, whereinthe method comprises: administering to the subject an effective amountof one or more miR-29s sufficient to decrease expression of at least onemethyltransferase comprising DNMT1, DNMT3A, and/or DNMT3B; and restoringexpression of the at least one TSG in the subject, wherein the restoringexpression of the at least one TSG includes inducing expression of theone or more miR-29s in the subject, wherein the one or more miR-29scomprise at least one isolated or synthetic miR-29 selected from thegroup consisting of miR-29a, miR-29b, and miR-29c, and wherein thesubject has a cancer having an increased expression of one or more ofDNMT1, DNMT3a and/or DNMT3B.
 37. The method of claim 36, wherein the atleast one TSG comprises FHIT and/or WWOX.
 38. A method for restoringexpression of at least one tumor suppression gene (TSG) in a cell inneed thereof, wherein the method comprises: transfecting the cell withan effective amount of one or more miR-29s sufficient to decreaseexpression of at least one methyltransferase comprising DNMT1, DNMT3A,and/or DNMT3B; and restoring expression of the at least one TSG in thecell, wherein the restoring expression of the at least one TSG includesinducing expression of the one or more miR-29s in the cell, wherein theone or more miR-29s comprise at least one isolated or synthetic miR-29selected from the group consisting of miR-29a, miR-29b, and miR-29c, andwherein the cell is a cancer cell having an increased expression of oneor more of DNMT1, DNMT3a and/or DNMT3B.
 39. The method of claim 38,wherein the at least one TSG comprises FHIT and/or WWOX.
 40. A methodfor developing an epigenetic therapy in a subject in need thereof,wherein the method comprises: administering to the subject an effectiveamount of one or more miR-29s sufficient to normalize an aberrantpattern of methylation and reactivate expression of at least one tumorsuppressor gene (TSG); and developing the epigenetic therapy in thesubject, wherein the one or more miR-29s comprise at least one isolatedor synthetic miR-29 selected from the group consisting of miR-29a,miR-29b, and miR-29c, and wherein the subject has a cancer having anincreased expression of one or more of DNMT1, DNMT3a and/or DNMT3B. 41.A method for developing an epigenetic therapy in a cell in need thereof,wherein the method comprises: transfecting the cell with an effectiveamount of one or more miR-29s sufficient to normalize an aberrantpattern of methylation and reactivate expression of at least one tumorsuppressor gene (TSG); and developing the epigenetic therapy in thecell, wherein the one or more miR-29s comprise at least one isolated orsynthetic miR-29 selected from the group consisting of miR-29a, miR-29b,and miR-29c, and wherein the cell is a cancer cell having an increasedexpression of one or more of DNMT1, DNMT3a and/or DNMT3B.